Method for producing ketocarotenoids by cultivating genetically modified organisms

ABSTRACT

The present invention relates to a process for preparing ketocarotenoids by cultivation of genetically modified organisms which, compared with the wild type, have a modified ketolase activity, to the genetically modified organisms, and to the use thereof as human and animal foods and for producing ketocarotenoid extracts.

A process for preparing ketocarotenoids by cultivation of geneticallymodified organisms

The present invention relates to a process for preparing ketocarotenoidsby cultivation of genetically modified organisms which, compared withthe wild type, have a modified ketolase activity, to the geneticallymodified organisms, and to the use thereof as human and animal foods andfor producing ketocarotenoid extracts.

Ketocarotenoids occur mainly in bacteria, a few fungi and as secondarycarotenoids in green algae. Besides echinenone, the 4-monoketoderivative of β-carotene, there is also formation of a correspondingsymmetric diketo compound canthaxanthin. In addition, a few species areknown of the abovementioned organism groups in which astaxanthin(=3,3′-dihydroxy-4,4′-diketo-β-carotene) is to be found as end productof biosynthesis (together with small amounts of correspondingintermediates) (Goodwin, T. W. (1980) The Biochemistry of theCarotenoids, Vol. 1: Plants, 2nd edn. Chapman & Hall, New York.;Johnson, E. A. & An G.-H. (1991) astaxanthin from microbial sources.Critical Rev. Biotechnol. 11, 297-326.; 3. Lorenz, R. T. & Cysewski, G.R. (2000) Commercial potential for Haematococcus microalgae as a naturalsource of astaxanthin. Trend Biotechn. 18, 160-167).

Because of their coloring properties, the ketocarotenoids and especiallyastaxanthin are used as pigmenting aids in livestock nutrition,especially in trout, salmon and shrimp rearing.

astaxanthin is currently prepared for the most part by chemicalsynthesis processes. Natural ketocarotenoids such as, for example,natural astaxanthin are currently obtained in small quantities inbiotechnological processes by cultivation of algae, for exampleHaematococcus pluvialis or by fermentation of genetically optimizedmicroorganisms and subsequent isolation.

An economic biotechnological process for preparing naturalketocarotenoids is therefore of great importance.

Specific ketolase genes of the crtW type have been cloned andfunctionally identified from the bacteria Agrobacterium aurantiacum (EP735 137, Accession No. D58420), Paracoccus marcusii (Accession No.Y15112) and as cDNA from Haematococcus (Haematococcus pluvialis Flotowem. Wille and Haematoccus pluvialis, NIES-144 (EP 725137, WO 98/18910and Lotan et al, FEBS Letters 1995, 364,125-128, Accession No. X86782and D45881)).

There also exist ORFs from other organisms which, because of amino acidhomologies, are referred to as ketolase genes, such as, for example,nucleic acids encoding a ketolase from Alcaligenes sp. PC-1 (EP 735137,Accession No. D58422), Synechocystis sp. strain PC6803 (Accession No.NP_(—)442491), Bradyrhizobium sp. (Accession No. AF218415), Nostoc sp.PCC 7120 (Kaneko et al, DNA Res. 2001, 8(5), 205-213; Accession No.AP003592, BAB74888) and Brevundimonas aurantiaca (WO 02079395).

Only the ketolases from A. aurantiacum and Alcaligenes spec. have beenbiochemically characterized (Fraser P. D., Shimada H. & Misawa N. (1998)Enzymic confirmation of reactions involved in routes to astaxanthinformation, elucidated using a direct substrate in vitro assay. Eur. J.Biochem. 252, 229-236.). There is a further type of 1-carotene ketolasegenes, crtO from the cyanobacterium Synechocystis, which has nosimilarity with crtW and is related to the bacterial desaturases(Femandez-Gonzalez, B., Sandmann, G. & Vioque, A (1997) A new type ofasymmetrically acting 1-carotene ketolase is required for the synthesisof echinenone in the cyanobacterium Synechocystis sp. PCC 6803. J. Biol.Chem. 272, 9728-9733.)

All known ketolases are able to introduce a keto group in position 4 ofβ-carotene. The crtO gene codes for a monoketolase which formsechinenone as end product from β-carotene. The crtW gene family, towhich bkt from Haematococcus also belongs, codes for a diketolase whichconverts β-carotene as far as canthaxanthin. This reaction appears to bethe first modification step in the direction of astaxanthin, which isfollowed by a hydroxylation at position 3. The same reaction sequencethen also applies to the second ionone ring (9). There is also enzymaticevidence that 3-hydroxy-β-carotene derivatives can be ketonized onlypoorly at position 4. It has likewise emerged that only certainbacterial hydroxylases, such as those from Erwinia uredovora(Breitenbach, J., Misawa, N., Kajiwara, S. & Sandmann, G. (1996)Expression in Escherichia coli and properties of the carotene ketolasefrom Haematococcus pluvialis. FEMS Microbiol. Left. 140, 241-246) or A.aurantiacum, are able to convert ketonized intermediates. Thestructurally different hydroxylases of cyanobacteria are not capable ofthis (Albrecht, M., Steiger, S. & Sandmann, G. (2001) Expression of aketolase gene mediates the synthesis of canthaxanthin in Synechococcusleading to resistance against pigment photodegradation and UV-Bsensitivity of photosynthesis. Photochem. Photobiol. 73, 551-555). Thereis no cooperation of this type of hydroxylase with a ketolase, and nosubstantial quantities of astaxanthin are obtained.

EP 735 137 describes the preparation of xanthophylls in microorganismssuch as, for example, E. coli by introducing ketolase genes (crtw) fromAgrobacterium aurantiacum or Alcaligenes sp. PC-1 into microorganisms.

EP 725 137, WO 98/18910, Kajiwara et al. (Plant Mol. Biol. 1995, 29,343-352) and Hirschberg et al. (FEBS Letters 1995, 364, 125-128)disclose the preparation of astaxanthin by introducing ketolase genesfrom Haematococcus pluvialis (crtW, crtO or bkt) into E. coli.

Hirschberg et al. (FEBS Letters 1997, 404, 129-134) describe thepreparation of astaxanthin in Synechococcus by introducing ketolasegenes (crtO) from Haematococcus pluvialis. Sandmann et al.(Photochemistry and Photobiology 2001, 73(5), 551-55) describe ananalogous process which, however, leads to the preparation ofcanthaxanthin and provides only traces of astaxanthin.

WO 98/18910 and Hirschberg et al. (Nature Biotechnology 2000, 18(8),888-892) describe the synthesis of ketocarotenoids in nectaries oftobacco flowers by introducing the ketolase gene from Haematococcuspluvialis (crtO) into tobacco.

WO 01/20011 describes a DNA construct for producing ketocarotenoids,especially astaxanthin, in seeds of oilseed crops such as rape,sunflower, soybean and mustard, using a seed-specific promoter and aketolase from Haematococcus pluvialis.

All the processes described in the prior art for preparingketocarotenoids and, in particular, the processes described forpreparing astaxanthin have the disadvantage that the transgenicorganisms provide only small quantities of astaxanthin.

It is an object of the present invention to provide a process forpreparing ketocarotenoids by cultivation of genetically modifiedorganisms, and to provide further genetically modified organisms whichproduce ketocarotenoids, which have the prior art disadvantagesdescribed above to a smaller extent or not at all.

We have found that this object is achieved by a process for preparingketocarotenoids by cultivating genetically modified organisms which,compared with the wild type, have a modified ketolase activity, and themodified ketolase activity is caused by a ketolase comprising the aminoacid sequence SEQ. ID. NO. 2 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhas an identity of at least 42% at the amino acid level with thesequence SEQ. ID. NO. 2.

The organisms of the invention, such as, for example, microorganisms orplants, are preferably able as starting organisms naturally to producecarotenoids such as, for example, β-carotene or zeaxanthin, or can bemade able by genetic modification such as, for example, reregulation ofmetabolic pathways or complementation to produce carotenoids such as,for example, β-carotene or zeaxanthin.

Some organisms are already able as starting or wild-type organisms toproduce ketocarotenoids such as, for example, astaxanthin orcanthaxanthin. These organisms, such as, for example, Haematococcuspluvialis, Paracoccus marcusil, Xanthophyllomyces dendrorhous, Bacilluscirculans, Chlorococcum, Phaffia rhodozyma, Adonis sp., Neochloriswimmeri, Protosiphon botryoides, Scotiellopsis oocystiformis,Scenedesmus vacuolatus, Chlorela zofingiensis, Ankistrodesmus braunii,Euglena sanguinea, Bacillus atrophaeus, Blakeslea already have asstarting or wild-type organism a ketolase activity.

In one embodiment of the process of the invention, therefore, thestarting organisms used are those already having a ketolase activity aswild type or starting organism. In this embodiment, the geneticmodification brings about an increase in the ketolase activity comparedwith the wild type or starting organism.

Ketolase activity means the enzymic activity of a ketolase.

A ketolase means a protein which has the enzymatic activity ofintroducing a keto group on the, optionally substituted, β-ionone ringof carotenoids.

A ketolase means in particular a protein having the enzymatic activityof converting β-carotene into canthaxanthin.

Accordingly, ketolase activity means the amount of β-carotene convertedor amount of canthaxanthin produced in a particular time by the ketolaseprotein.

Thus, when a ketolase activity is increased compared with the wild type,the amount of β-carotene converted or the amount of canthaxanthinproduced in a particular time is increased by the ketolase proteincompared with the wild type.

This increase in the ketolase activity is preferably at least 5%, morepreferably at least 20%, more preferably at least 50%, more preferablyat least 100%, preferably at least 300%, more preferably at least 500%,in particular at least 600%, of the ketolase acitivty of the wild type.

The term “wild type” means according to the invention the correspondingstarting organism.

Depending on the context, the term “organism” may mean the startingorganism (wild type) or a genetically modified organism of theinvention, or both.

“Wild type” means, preferably and especially in cases where the organismor the wild type cannot be unambiguously assigned, in each case areference organism for the increasing or causing of the ketolaseactivity, for the increasing, described hereinafter, of the hydroxylaseactivity, for the increasing, described hereinafter, of the β-cyclaseactivity and the increasing of the content of ketocarotenoids.

This reference organism for microorganisms which already have a ketolaseactivity as wild type is preferably Haematococcus pluvialis.

This reference organism for microorganisms which have no ketolaseactivity as wild type is preferably Blakeslea.

This reference organism for plants which already have a ketolaseactivity as wild type is preferably Adonis aestivalis, Adonis flammeusor Adonis annuus, particularly preferably Adonis aestivalis.

This reference organism for plants which have no ketolase activity inpetals as wild type is preferably Tagetes erecta, Tagetes patula,Tagetes lucida, Tagetes pringlei, Tagetes palmeri, Tagetes minuta orTagetes campanulata, particularly preferably Tagetes erecta.

Determination of the ketolase activity in the genetically modifiedorganisms of the invention and in wild-type and reference organismspreferably takes place under the following conditions:

Determination of the ketolase activity in plant or microorganismmaterial is based on the method of Frazer et al., (J. Biol. Chem.272(10): 6128-6135, 1997). The ketolase activity in plant ormicroorganism extracts is determined using the substrates β-carotene andcanthaxanthin in the presence of lipid (soybean lecithin) and detergent(sodium cholate). Substrate/product ratios from ketolase assays aremeasured by means of HPLC.

Various ways are possible for increasing the ketolase activity, forexample by switching off inhibitory regulatory mechanisms at thetranslation and protein level or by increasing the gene expression of anucleic acid encoding a ketolase compared with the wild type, forexample by inducing the ketolase gene by activators or by introducingnucleic acids encoding a ketolase into the organism.

Increasing the gene expression of a nucleic acid encoding a ketolasealso means according to the invention in this embodiment themanipulation of the expression of the organisms own endogenousketolases. This can be achieved for example by modifying the promoterDNA sequence for ketolase-encoding genes. Such a modification, whichresults in a modified or, preferably, increased expression rate of atleast one endogenous ketolase gene, can also be effected by deletion orinsertion of DNA sequences.

It is possible as described above to modify the expression of at leastone endogenous ketolase through application of exogenous stimuli. Thiscan be effected by particular physiological conditions, i.e. throughapplication of foreign substances.

A further possibility for achieving an increased expression of at leastone endogenous ketolase gene is for a regulator protein which does notoccur in the wild-type organism or is modified to interact with thepromoter of these genes.

A regulator of this type may be a chimeric protein which consists of aDNA-binding domain and of a transcription activator domain as described,for example, in WO 96/06166.

In a preferred embodiment, the ketolase activity is increased bycomparison with the wild type by increasing the gene expression of anucleic acid encoding a ketolase comprising the amino acid sequence SEQ.ID. NO. 2 or a sequence which is derived from this sequence bysubstitution, insertion or deletion of amino acids and which has anidentity of at least 42% at the amino acid level with the sequence SEQ.ID. NO. 2.

In a further preferred embodiment, the gene expression of a nucleic acidencoding a ketolase is increased by introducing nucleic acids whichencode ketolases, where the ketolases have the amino acid sequence SEQ.ID. NO. 2 or a sequence which is derived from this sequence bysubstitution, insertion or deletion of amino acids and which has anidentity of at least 42% at the amino acid level with the sequence SEQ.ID. NO. 2, into the organisms.

Thus, in this embodiment, at least one further ketolase gene encoding aketolase comprising the amino acid sequence SEQ. ID. NO. 2 or a sequencewhich is derived from this sequence by substitution, insertion ordeletion of amino acids and which has an identity of at least 42% at theamino acid level with the sequence SEQ. ID. NO. 2, is present in thetransgenic organisms of the invention compared with the wild type.

In this embodiment, the genetically modified organism of the inventionaccordingly has at least one exogenous (=heterologous) nucleic acidencoding a ketolase, or has at least two endogenous nucleic acidsencoding a ketolase, where the ketolases comprise the amino acidsequence SEQ. ID. NO. 2 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhas an identity of at least 42% at the amino acid level with thesequence SEQ. ID. NO. 2.

In another, preferred embodiment of the process of the invention, theorganisms used as starting organisms have no ketolase activity as wildtype.

In this preferred embodiment, the genetic modification causes theketolase activity in the organisms. The genetically modified organism ofthe invention thus has in this preferred embodiment a ketolase activitycompared with the genetically unmodified wild type, and is thuspreferably capable of transgenic expression of a ketolase comprising theamino acid sequence SEQ. ID. NO. 2 or a sequence which is derived fromthis sequence by substitution, insertion or deletion of amino acids andwhich has an identity of at least 42% at the amino acid level with thesequence SEQ. ID. NO. 2.

In this preferred embodiment, the gene expression of a nucleic acidencoding a ketolase is caused, in analogy to the increasing, describedabove, of the gene expression of a nucleic acid encoding a ketolase,preferably by introducing nucleic acids which encode ketolasescomprising the amino acid sequence SEQ. ID. NO. 2 or a sequence which isderived from this sequence by substitution, insertion or deletion ofamino acids and which has an identity of at least 42% at the amino acidlevel with the sequence SEQ. ID. NO. 2, into the starting organism.

It is possible to use for this purpose in both embodiments in principleall nucleic acids which encode a ketolase comprising the amino acidsequence SEQ. ID. NO. 2 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhas an identity of at least 42% at the amino acid level with thesequence SEQ. ID. NO. 2.

The use of the nucleic acids of the invention encoding a ketolase leadsin the process of the invention surprisingly to a higher yield ofketocarotenoids, especially of astaxanthin, than on use of the ketolasegenes used in the prior art.

All the nucleic acids mentioned in the description may be, for example,an RNA, DNA or cDNA sequence.

In the case of genomic ketolase sequences from eukaryotic sources whichcomprise introns, it is preferred to use nucleic acid sequences whichhave already been processed, such as the corresponding cDNAs, in thecase where the host organism is unable or cannot be made able to expressthe corresponding ketolase.

Examples of nucleic acids encoding a ketolase, and the correspondingketolases comprising the amino acid sequence SEQ. ID. NO. 2 or asequence which is derived from this sequence by substitution, insertionor deletion of amino acids and which has an identity of at least 42% atthe amino acid level with the sequence SEQ. ID. NO. 2, which can be usedadvantageously in the process of the invention are, for example,sequences from

Nostoc punctiforme PCC73102 ORF 38, nucleic acid: Acc. No.NZ_AABC01000195, base pair 55,604 to 55,392 (SEQ ID NO: 1); protein:Acc. No. ZP_(—)00111258 (SEQ ID NO: 2) (annotated as putative protein)or

Nostoc punctiforme PCC73102 ORF 148, nucleic acid: Acc. No.NZ_AABC01000196, base pair 140,571 to 139,810 (SEQ ID NO: 3), protein:(SEQ ID NO: 4) (not annotated) or ketolase sequences derived from thesesequences.

FIG. 1 shows additionally the nucleic acid sequences of ORF 38 and ORF148 from Nostoc punctiforme.

For the preparation of astaxanthin it is particularly preferred to usein particular the ketolase of Nostoc punctiforme PCC73102 ORF 148,nucleic acid: Acc. No. NZ_AABC01000196, base pair 140,571 to 139,810(SEQ ID NO: 3), protein: (SEQ ID NO: 4) or sequences derived from thissequence.

Further natural examples of ketolases and ketolase genes which can beused in the process of the invention can easily be found for examplefrom various organisms whose genomic sequence is known through identitycomparisons of the amino acid sequences or of the correspondingback-translated nucleic acid sequences from databases with the sequencesSEQ ID NO: 2 or SEQ ID NO: 4 described above.

Further natural examples of ketolases and ketolase genes canadditionally be easily found starting from the nucleic acid sequencesabove, in particular starting from the sequences SEQ ID NO: 1 or SEQ IDNO: 3 from various organisms whose genomic sequence is unknown throughhybridization techniques in a manner known per se.

The hybridization can take place under moderate (low stringency) orpreferably under stringent (high stringency) conditions.

Hybridization conditions of these types are described for example inSambrook, J., Fritsch, E. F., Maniatis, T., in: Molecular Cloning (ALaboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press,1989, pages 9.31-9.57 or in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

For example, the conditions during the washing step can be selected fromthe range of conditions limited by those of low stringency (with 2×SSCat 50° C.) and those of high stringency (with 0.2×SSC at 50° C.,preferably at 65° C.) (20×SSC: 0.3 M sodium citrate, 3 M sodiumchloride, pH 7.0).

An additional possibility is to raise the temperature during the washingstep from moderate conditions at room temperature, 22° C., up tostringent conditions at 65° C.

Both parameters, the salt concentration and temperature, can be variedsimultaneously, and it is also possible to keep one of the twoparameters constant and vary only the other one. It is also possible toemploy denaturing agents such as, for example, formamide or SDS duringthe hybridization. Hybridization in the presence of 50% formamide ispreferably carried out at 42° C.

Some examples of conditions for hybridization and washing step are givenbelow:

(1) Hybridization Conditions with for Example

-   (i) 4×SSC at 65° C., or-   (ii) 6×SSC at 45° C., or-   (iii) 6×SSC at 68° C., 100 mg/ml denatured fish sperm DNA, or-   (iv) 6×SSC, 0.5% SDS, 100 mg/ml denatured, fragmented salmon sperm    DNA at 68° C., or-   (v) 6×SSC, 0.5% SDS, 100 mg/ml denatured, fragmented salmon sperm    DNA, 50% formamide at 42° C., or-   (vi) 50% formamide, 4×SSC at 42° C., or-   (vii) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1%    Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH    6.5, 750 mM NaCl, 75 mM sodium citrate at 42° C., or-   (viii) 2× or 4×SSC at 50° C. (moderate conditions), or-   (ix) 30 to 40% formamide, 2× or 4×SSC at 42° C. (moderate    conditions).    (2) Washing Step for 10 Minutes Each with for Example-   (i) 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C., or-   (ii) 0.1×SSC at 65° C., or-   (iii) 0.1×SSC, 0.5% SDS at 68° C., or-   (iv) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C., or-   (v) 0.2×SSC, 0.1% SDS at 42° C., or-   (vi) 2×SSC at 65° C. (moderate conditions).

In a preferred embodiment of the process of the invention there isintroduction of nucleic acids which encode a ketolase comprising theamino acid sequence SEQ ID NO: 2 or a sequence which is derived fromthis sequence by substitution, insertion or deletion of amino acids andwhich has an identity of at least 50%, preferably at least 60%,preferably at least 65%, preferably at least 70%, more preferably atleast 75%, more preferably at least 80%, more preferably at least 85%,more preferably at least 90%, more preferably at least 95%, particularlypreferably at least 98%, at the amino acid level with the sequence SEQID NO: 2.

It is moreover possible for the ketolase sequence to be a natural onewhich can be found as described above by identity comparison of thesequences from other organisms, or for the ketolase sequence to be anartificial one which has been modified starting from the sequence SEQ IDNO: 2 by artificial variation, for example by substitution, insertion ordeletion of amino acids.

The term “substitution” means in the description substitution of one ormore amino acids by one or more amino acids. So-called conservativesubstitutions are preferably carried out, in which the replaced aminoacid has a similar property to the original amino acid, for examplesubstitution of Glu by Asp, Gln by Asn, Val by lie, Leu by lIe, Ser byThr.

Deletion is the replacement of an amino acid by a direct linkage.Preferred positions for deletions are the termini of the polypeptide andthe linkages between the individual protein domains.

Insertions are introductions of amino acids into the polypeptide chain,with formal replacement of a direct linkage by one or more amino acids.

Identity between two proteins means the identity of the amino acids overthe entire length of each protein, in particular the identity calculatedby comparison using the vector NTI suite 7.1 software supplied byInformax (USA) using the clustal method (Higgins D G, Sharp P M. Fastand sensitive multiple sequence alignments on a microcomputer. ComputAppl. Biosci. 1989 Apr.; 5(2):151-1), setting the following parameters:

Multiple Alignment Parameter: Gap opening penalty 10 Gap extensionpenalty 10 Gap separation penalty range 8 Gap separation penalty off %identity for alignment delay 40 Residue specific gaps off Hydrophilicresidue gap off Transition weight 0

Pairwise Alignment Parameter: FAST algorithm on K-tuple size 1 Gappenalty 3 Window size 5 Number of best diagonals 5

The ketolase having an identity of at least 42% at the amino acid levelwith the sequence SEQ ID NO: 2 accordingly means a ketolase which, oncomparison of its sequence with the sequence SEQ ID NO: 2, in particularusing the above program algorithm with the above set of parameters, hasan identity of at least 42%.

For example, using the above program algorithm with the above set ofparameters, the sequence of the ketolase from Nostoc punctiformePCC73102 ORF 148 (SEQ ID NO: 4) shows an identity of 64% with thesequence of the ketolase from Nostoc punctiforme PCC73102 ORF 38 (SEQ IDNO: 2).

Suitable nucleic acid sequences can be obtained for example byback-translation of the polypeptide sequence in accordance with thegenetic code.

The codons preferably used for this purpose are those frequently used inaccordance with the organism-specific codon usage. The codon usage caneasily be found by means of computer analyses of other, known genes inthe relevant organisms.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ ID NO: 1 or SEQ ID NO: 3 is introduced into the organism.

All the aforementioned ketolase genes can moreover be prepared in amanner known per se by chemical synthesis from the nucleotide units suchas, for example, by fragment condensation of individual overlapping,complementary nucleic acid units of the double helix. Chemical synthesisof oligonucleotides is possible, for example, in a known manner by thephosphoramidite method (Voet, Voet, 2nd edition, Wiley Press New York,pages 896-897). Addition of synthetic oligonucleotides and filling in ofgaps using the Klenow fragment of DNA polymerase and ligation reactions,and general cloning methods, are described in Sambrook et al. (1989),Molecular cloning: A laboratory manual, Cold Spring Harbor LaboratoryPress.

The identity shown by the sequence of the ketolase from Nostocpunctiforme PCC73102 ORF 38 (SEQ ID NO: 2) with the sequences of theketolases used in the prior art processes is 38% (Agrobacteriumaurantiacum (EP 735 137), Accession No. D58420), 38% (Alcaligenes sp.PC-1 (EP 735137), Accession No. D58422) and 19 to 21% (Haematococcuspluvialis Flotow em. Wille and Haematoccus pluvialis, NIES 144 (EP725137, WO 98/18910 and Lotan et al, FEBS Letters 1995, 364, 125 128),Accession No. X86782 and D45881).

In a preferred embodiment, organisms which have an increased hydroxylaseactivity and/or β-cyclase activity in addition to the increased ketolaseactivity compared with the wild type are cultivated.

Hydroxylase activity means the enzymic activity of a hydroxylase.

A hydroxylase means a protein having the enzymatic activity ofintroducing a hydroxyl group on the, optionally substituted, β-iononering of carotenoids.

In particular, a hydroxylase means a protein having the enzymaticactivity of converting β-carotene into zeaxanthin or canthaxanthin intoastaxanthin.

Accordingly, hydroxylase activity means the amount of β-carotene orcanthaxanthin converted, or amount of zeaxanthin or astaxanthinproduced, by the hydroxylase protein.

Thus, when the hydroxylase activity is increased compared with the wildtype, the amount of β-carotene or canthaxantin converted or the amountof zeaxanthin or astaxanthin produced in a particular time by thehydroxylase protein is increased compared with the wild type.

This increase in the hydroxylase activity is preferably at least 5%,further preferably at least 20%, further preferably at least 50%,further preferably at least 100%, more preferably at least 300%, evenmore preferably at least 500%, in particular at least 600%, of thehydroxylase activity of the wild type.

β-Cyclase activity means the enzymic activity of a β-cyclase.

A β-cyclase means a protein having the enzymatic activity of convertinga terminal linear lycopene residue into a β-ionone ring.

In particular, a β-cyclase means a protein having the enzymatic activityof converting γ-carotene into β-carotene.

Accordingly, a β-cyclase activity means the amount of γ-caroteneconverted or the amount of β-carotene produced in a particular time bythe β-cyclase protein.

Thus, when the β-cyclase activity is increased compared with the wildtype, the amount of lycopene or γ-carotene converted or the amount ofγ-carotene produced from lycopene or the amount of β-carotene producedfrom carotene by the β-cyclase protein in a particular time is increasedcompared with the wild type.

This increase in the β-cyclase activity is preferably at least 5%,further preferably at least 20%, further preferably at least 50%,further preferably at least 100%, more preferably at least 300%, evenmore preferably at least 500%, in particular at least 600%, of theβ-cyclase activity of the wild type.

The hydroxylase activity in the genetically modified organisms of theinvention and in wild-type and reference organisms is preferablydetermined under the following conditions:

The hydroxylase activity is determined by the method of Bouvier et al.(Biochim. Biophys. Acta 1391 (1998), 320-328) in vitro. Ferredoxin,ferredoxin-NADP⁺ oxidoreductase, catalase, NADPH and β-carotene withmono- and digalactosyl glycerides are added to a defined amount oforganism extract.

The hydroxylase activity is particularly preferably determined under thefollowing conditions of Bouvier, Keller, d'Harlingue and Camara(Xanthophyll biosynthesis: molecular and functional characterization ofcarotenoid hydroxylases from pepper fruits (Capsicum annuum L.);Biochim. Biophys. Acta 1391 (1998), 320-328):

The in vitro assay is carried out in a volume of 0.250 ml. The mixturecontains 50 mM potassium phosphate (pH 7.6), 0.025 mg of spinachferredoxin, 0.5 units of spinach ferredoxin-NADP⁺ oxidoreductase, 0.25mM NADPH, 0.010 mg of beta-carotene (emulsified in 0.1 mg of Tween 80),0.05 mM of a mixture of mono- and digalactosyl glycerides (1:1), 1 unitof catalyse, 0.2 mg of bovine serum albumin and organism extract in adifferent volume. The reaction mixture is incubated at 30° C. for 2hours. The reaction products are extracted with organic solvents such asacetone or chloroform/methanol (2:1) and determined by HPLC.

The β-cyclase activity in the genetically modified organisms of theinvention and in wild-type and reference organisms is preferablydetermined under the following conditions:

The β-cyclase activity is determined by the method of Fraser andSandmann (Biochem. Biophys. Res. Comm. 185(1) (1992) 9 15) in vitro.Potassium phosphate is added as buffer (pH 7.6), lycopene as substrate,paprika stromal protein, NADP⁺, NADPH and ATP to a defined amount oforganism extract.

The β-cyclase activity is particularly preferably determined under thefollowing conditions of Bouvier, d'Harlingue and Camara (MolecularAnalysis of carotenoid cyclase inhibition; Arch. Biochem. Biophys.346(1) (1997) 53-64):

The in vitro assay is carried out in a volume of 250 μl. The mixturecontains 50 mM potassium phosphate (pH 7.6), various amounts of organismextract, 20 nM lycopene, 250 μg of paprika chromoplastid stromalprotein, 0.2 mM NADP⁺, 0.2 mM NADPH and 1 mM ATP. NADP/NADPH and ATP aredissolved in 10 ml of ethanol with 1 mg of Tween 80 immediately beforeaddition to the incubation medium. After a reaction time of 60 minutesat 30° C., the reaction is stopped by adding chloroform/methanol (2:1).The reaction products extracted into chloroform are analyzed by HPLC.

An alternative assay with radioactive substrate is described in Fraserand Sandmann (Biochem. Biophys. Res. Comm. 185(1) (1992) 9-15).

The hydroxylase activity and/or β-cyclase activity can be increased invarious ways, for example by switching off inhibitory regulatorymechanisms at the expression and protein level or by increasing the geneexpression of nucleic acids encoding a hydroxylase, and/or of nucleicacids encoding a β-cyclase, compared with the wild type.

The gene expression of nucleic acids encoding a hydroxylase, and/or thegene expression of the nucleic acid encoding a β-cyclase, compared withthe wild type, can likewise be increased in various ways, for example byinducing the hydroxylase gene and/or β-cyclase gene by activators or byintroducing one or more hydroxylase gene copies and/or β-cyclase genecopies, i.e. by introducing at least one nucleic acid encoding ahydroxylase, and/or at least one nucleic acid encoding a β-cyclase, intothe organism.

Increasing the gene expression of a nucleic acid encoding a hydroxylaseand/or β-cyclase also means according to the invention manipulation ofthe expression of the organism's own endogenous hydroxylase and/orβ-cyclase.

This can be achieved for example by modifying the promoter DNA sequencefor genes encoding hydroxylases and/or β-cyclases. Such a modification,resulting in an increased expression rate of the gene, can be effectedfor example by deletion or insertion of DNA sequences.

It is possible, as described above, to modify the expression of theendogenous hydroxylase and/or β-cyclase by application of exogenousstimuli. This can be effected by particular physiological conditions,i.e. by application of foreign substances.

A further possibility for achieving a modified or increased expressionof an endogenous hydroxylase and/or β-cyclase gene is throughinteraction of a regulator protein which does not occur in theuntransformed organism with the promoter of this gene.

Such a regulator may be a chimeric protein consisting of a DNA-bindingdomain and of a transcription activator domain as described, forexample, in WO 96/06166.

In a preferred embodiment, the gene expression of a nucleic acidencoding a hydroxylase, and/or the gene expression of a nucleic acidencoding a β-cyclase, is increased by introducing at least one nucleicacid encoding a hydroxylase, and/or by introducing at least one nucleicacid encoding a β-cyclase, into the organism.

It is possible to use for this purpose in principle any hydroxylase geneor any β-cyclase gene, i.e. any nucleic acid which encodes a hydroxylaseand any nucleic acid which encodes a β-cyclase.

In the case of genomic hydroxylase or β-cyclase nucleic acid sequencesfrom eukaryotic sources which comprise introns, it is preferred to usenucleic acid sequences which have already been processed, such as thecorresponding cDNAs, in the case where the host organism is unable orcannot be made able to express the corresponding hydroxylase orβ-cyclase.

One example of a hydroxylase gene is a nucleic acid encoding ahydroxylase from Haematococcus pluvialis (Accession AX038729, WO0061764); (nucleic acid: SEQ ID NO: 5, protein: SEQ ID NO: 6).

One example of a β-cyclase gene is a nucleic acid encoding a α-cyclasefrom tomato (Accession X86452) (nucleic acid: SEQ ID NO: 7, protein: SEQID NO: 8).

Thus, in this preferred embodiment, at least one further hydroxylasegene and/or β-cyclase gene is present in the preferred transgenicorganisms of the invention compared with the wild type.

In this preferred embodiment, the genetically modified organism has forexample at least one exogenous nucleic acid encoding a hydroxylase, orat least two endogenous nucleic acids encoding a hydroxylase and/or atleast one exogenous nucleic acid encoding a β-cyclase, or at least twoendogenous nucleic acids encoding a α-cyclase.

The hydroxylase genes preferably used in the preferred embodimentdescribed above are nucleic acids encoding proteins comprising the aminoacid sequence SEQ ID NO: 6 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhave an identity of at least 30%, preferably at least 50%, morepreferably at least 70%, even more preferably at least 90%, mostpreferably at least 95%, at the amino acid level with the sequence SEQID NO: 6, and which have the enzymatic property of a hydroxylase.

Further examples of hydroxylases and hydroxylase genes can be easilyfound for example from various organisms whose genomic sequence is knownas described above by homology comparisons of the amino acid sequencesor of the corresponding back-translated nucleic acid sequences fromdatabases with SEQ ID. NO: 6.

Further examples of hydroxylases and hydroxylase genes can easily befound in a manner known per se in addition for example starting from thesequence SEQ ID NO: 5 from various organisms whose genomic sequence isunknown, as described above, by hybridization and PCR techniques.

In a further particularly preferred embodiment, nucleic acids whichencode proteins comprising the amino acid sequence of the hydroxylase ofthe sequence SEQ ID NO: 6 are introduced into organisms to increase thehydroxylase activity.

Suitable nucleic acid sequences can be obtained for example byback-translation of the polypeptide sequence in accordance with thegenetic code.

The codons used for this purpose are preferably those frequently used inaccordance with the organism-specific codon usage. This codon usage caneasily be found by means of computer analyses of other, known genes ofthe relevant organisms.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO: 5 is introduced into the organism.

The β-cyclase genes preferably used in the preferred embodimentdescribed above are nucleic acids which encode proteins comprising theamino acid sequence SEQ ID NO: 8 or a sequence which is derived fromthis sequence by substitution, insertion or deletion of amino acids andwhich has an identity of at least 30%, preferably at least 50%, morepreferably at least 70%, even more preferably at least 90%, mostpreferably at least 95%, at the amino acid level with the sequence SEQID NO: 8, and which has the enzymatic property of a β-cyclase.

Further examples of β-cyclases and β-cyclase genes can easily be foundfor example from various organisms whose genomic sequence is known asdescribed above by homology comparisons of the amino acid sequences orof the corresponding back-translated nucleic acid sequences fromdatabases with the SEQ ID NO: 8.

Further examples of β-cyclases and β-cyclase genes can easily be foundin a manner known per se in addition for example starting from thesequence SEQ ID NO: 7 from various organisms whose genomic sequence isunknown by hybridization and PCR techniques.

In a further particularly preferred embodiment, nucleic acids whichencode proteins comprising the amino acid sequence of β-cyclase of thesequence SEQ. ID. NO: 8 are introduced into organisms to increase theβ-cyclase activity.

Suitable nucleic acid sequences can be obtained for example byback-translation of the polypeptide sequence in accordance with thegenetic code.

The codons preferably used for this purpose are those frequently used inaccordance with the organ-specific codon usage. This codon usage caneasily be found by means of computer analyses of other, known genes ofthe relevant organisms.

In a particularly preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO: 7 is introduced into the organism.

All the aforementioned hydroxylase genes or β-cyclase genes can moreoverbe prepared in a manner known per se by chemical synthesis from thenucleotide units such as, for example, by fragment condensation ofindividual overlapping, complementary nucleic acid units of the doublehelix. Chemical synthesis of oligonucleotides is possible, for example,in a known manner by the phosphoramidite method (Voet, Voet, 2ndedition, Wiley Press New York, pages 896-897). Addition of syntheticoligonucleotides and filling in of gaps using the Klenow fragment of DNApolymerase and ligation reactions, and general cloning methods, aredescribed in Sambrook et al. (1989), Molecular cloning: A laboratorymanual, Cold Spring Harbor Laboratory Press.

The genetically modified organisms particularly preferably used in theprocess of the invention have the following combinations of geneticmodifications:

-   genetically modified organisms which have, compared with the wild    type, an increased or caused ketolase activity and an increased    hydroxylase activity,-   genetically modified organisms which have, compared with the wild    type, an increased or caused ketolase activity and an increased    β-cyclase activity and genetically modified organisms which have,    compared with the wild type, an increased or caused ketolase    activity and an increased hydroxylase activity and an increased    β-cyclase activity.

These genetically modified organisms can be produced as describedhereinafter for example by introducing individual nucleic acidconstructs (expression cassettes) or by introducing multiple constructswhich comprise up to two or three of the described activities.

Organisms preferably mean according to the invention organisms which areable as wild-type or starting organisms naturally or through geneticcomplementation and/or reregulation of metabolic pathways to producecarotenoids, in particular β-carotene and/or zeaxanthin and/orneoxanthin and/or violaxanthin and/or lutein.

Further preferred organisms already have as wild-type or startingorganisms a hydroxylase activity and are thus able as wild-type orstarting organisms to produce zeaxanthin.

Preferred organisms are plants or microorganisms such as, for example,bacteria, yeasts, algae or fungi.

Bacteria which can be used are both bacteria which are able, because ofthe introduction of genes of carotenoid biosynthesis of acarotenoid-producing organism, to synthesize xanthophylls, such as, forexample, bacteria of the genus Escherichia, which comprise for examplecrt genes from Erwinia, and bacteria which are intrinsically able tosynthesize xanthophylls, such as, for example, bacteria of the genusErwinia, Agrobacterium, Flavobacterium, Alcaligenes, Paracoccus, Nostocor cyanobacteria of the genus Synechocystis.

Preferred bacteria are Escherichia coli, Erwinia herbicola, Erwiniauredovora, Agrobacterium aurantiacum, Alcaligenes sp. PC-1,Flavobacterium sp. strain R1534, the cyanobacterium Synechocystis sp.PCC6803, Paracoccus marcusii or Paracoccus carotinifaciens.

Preferred yeasts are Candida, Saccharomyces, Hansenula, Pichia orPhaffia. Particularly preferred yeasts are Xanthophyllomyces dendrorhousor Phaffia rhodozyma.

Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora,Blakeslea, Phycomyces, Fusarium or other fungi described in Indian Chem.Engr. Section B. Vol. 37, No. 1, 2 (1995) on page 15, table 6.

Preferred algae are green algae such as, for example, algae of the genusHaematococcus, Phaedactylum tricornatum, Volvox or Dunaliella.Particularly preferred algae are Haematococcus pluvialis or Dunaliellabardawil.

Further microorganisms which can be used and the production thereof forcarrying out the process of the invention are disclosed for example inDE-A-199 16 140, which is incorporated herein by reference.

Particularly preferred plants are plants selected from the familiesRanunculaceae, Berberidaceae, Papaveraceae, Cannabaceae, Rosaceae,Fabaceae, Linaceae, Vitaceae, Brassicaceae, Cucurbitaceae, Primulaceae,Caryophyllaceae, Amaranthaceae, Gentianaceae, Geraniaceae,Caprifoliaceae, Oleaceae, Tropaeolaceae, Solanaceae, Scrophulariaceae,Asteraceae, Liliaceae, Amaryllidaceae, Poaceae, Orchidaceae, Malvaceae,llliaceae or Lamiaceae.

Very particularly preferred plants are selected from the group of plantgenera Marigold, Tagetes erecta, Tagetes patula, Acacia, Aconitum,Adonis, Amica, Aquilegia, Aster, Astragalus, Bignonia, Calendula,Caltha, Campanula, Canna, Centaurea, Cheiranthus, Chrysanthemum, Citrus,Crepis, Crocus, Curcurbita, Cytisus, Delonia, Delphinium, Dianthus,Dimorphotheca, Doronicum, Eschscholtzia, Forsythia, Fremontia, Gazania,Gelsemium, Genista, Gentiana, Geranium, Gerbera, Geum, Grevillea,Helenium, Helianthus, Hepatica, Heracleum, Hibiscus, Heliopsis,Hypericum, Hypochoeris, Impatiens, Iris, Jacaranda, Kenia, Labumum,Lathyrus, Leontodon, Lilium, Linum, Lotus, Lycopersicon, Lysimachia,Maratia, Medicago, Mimulus, Narcissus, Oenothera, Osmanthus, Petunia,Photinia, Physalis, Phyteuma, Potentilla, Pyracantha, Ranunculus,Rhododendron, Rosa, Rudbeckia, Senecio, Silene, Silphium, Sinapsis,Sorbus, Spartium, Tecoma, Torenia, Tragopogon, Trollius, Tropaeolum,Tulipa, Tussilago, Ulex, Viola or Zinnia, particularly preferablyselected from the group of plant genera Marigold, Tagetes erecta,Tagetes patula, Lycopersicon, Rosa, Calendula, Physalis, Medicago,Helianthus, Chrysanthemum, Aster, Tulipa, Narcissus, Petunia, Geranium,Tropaeolum or Adonis.

In the process of the invention for preparing ketocarotenoids, the stepof cultivating the genetically modified organisms is preferably followedby a harvesting of the organisms and further preferably in addition byan isolation of ketocarotenoids from the organisms.

The harvesting of the organisms takes place in a manner known per seappropriate for the particular organism. Microorganisms such asbacteria, yeasts, algae or fungi or plant cells cultivated byfermentation in liquid nutrient media can be removed for example bycentrifugation, decantation or filtration. Plants are grown on nutrientmedia and appropriately harvested in a manner known per se.

The genetically modified microorganisms are preferably cultivated in thepresence of oxygen at a cultivation temperature of at least about 20°C., such as for example, 20° C. to 40° C., and at a pH of about 6 to 9.In the case of genetically modified microorganisms, the microorganismsare preferably initially cultivated in the presence of oxygen and in acomplex medium such as, for example, TB or LB medium at a cultivationtemperature of about 20° C. or more, and at a pH of about 6 to 9, untila sufficient cell density is reached. In order to be able to control theoxidation reaction better, it is preferred to use an inducible promoter.The cultivation is continued after induction of ketolase expression inthe presence of oxygen for example for 12 hours to 3 days.

The ketocarotenoids are isolated from the harvested biomass in a mannerknown per se, for example by extraction and, where appropriate, furtherchemical or physical purification processes such as, for example,precipitation methods, crystallography, thermal separation processes,such as rectification processes or physical separation processes suchas, for example, chromatography.

As mentioned below, the ketocarotenoids can be specifically produced inthe genetically modified plants of the invention preferably in variousplant tissues such as, for example, seeds, leaves, fruits, flowers,especially in petals.

Ketocarotenoids are isolated from the harvested petals in a manner knownper se, for example by drying and subsequent extraction and, whereappropriate, further chemical or physical purification processes suchas, for example, precipitation methods, crystallography, thermalseparation processes such as rectification processes or physicalseparation processes such as, for example, chromatography.Ketocarotenoids are isolated from petals for example preferably byorganic solvents such as acetone, hexane, ether or methyl tert-butylether.

Further processes for isolating ketocarotenoids, especially from petals,are described for example in Egger and Kleinig (Phytochemistry (1967) 6,437-440) and Egger (Phytochemistry (1965) 4, 609-618).

The ketocarotenoids are preferably selected from the group ofastaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone,3′-hydroxyechinenone, adonirubin and adonixanthin.

astaxanthin is a particularly preferred ketocarotenoid.

Depending on the organism used, the ketocarotenoids are obtained in freeform or as fatty acid ester.

In plant petals, the ketocarotenoids are obtained in the process of theinvention in the form of their mono- or diesters with fatty acids. Someexamples of detected fatty acids are myristic acid, palmitic acid,stearic acid, oleic acid, linolenic acid and lauric acid (Kamata andSimpson (1987) Comp. Biochem. Physiol. Vol. 86B(3), 587-591).

The ketocarotenoids can be produced in the whole plant or, in apreferred embodiment, specifically in plant tissues containingchromoplasts. Examples of preferred plant tissues are roots, seeds,leaves, fruits, flowers and, in particular nectaries and petals.

In a particularly preferred embodiment of the process of the invention,genetically modified plants which show the highest rate of expression ofa ketolase in flowers are used.

This is preferably achieved through the ketolase gene expression beingunder the control of a flower-specific promoter. For this purpose, forexample, the nucleic acids described above are introduced into theplant, as described in detail below, in a nucleic acid constructfunctionally linked to a flower-specific promoter.

In a further, particularly preferred embodiment of the process of theinvention, genetically modified plants which show the highest rate ofexpression of a ketolase in fruits are used.

This is preferably achieved through the ketolase gene expression beingunder the control of a fruit-specific promoter. For this purpose, forexample, the nucleic acids described above are introduced into theplant, as described in detail below, in a nucleic acid constructfunctionally linked to a fruit-specific promoter.

In a further, particularly preferred embodiment of the process of theinvention, genetically modified seeds which show the highest rate ofexpression of a ketolase in seeds are used.

This is preferably achieved through the ketolase gene expression beingunder the control of a seed-specific promoter. For this purpose, forexample, the nucleic acids described above are introduced into theplant, as described in detail below, in a nucleic acid constructfunctionally linked to a seed-specific promoter.

The targeting into the chromoplasts is effected by a functionally linkedplastid transit peptide.

The production of genetically modified plants with increased or causedketolase activity is described by way of example below. Furtheractivities such as, for example, the hydroxylase activity and/or theβ-cyclase activity can be increased analogously using nucleic acidsequences encoding a hydroxylase or β-cyclase in place of nucleic acidsequences encoding a ketolase. The transformation can be effected in thecombinations of genetic modifications singly or by multiple constructs.

The transgenic plants are preferably produced by transformation of thestarting plants using a nucleic acid construct which comprises thenucleic acids described above encoding a ketolase, which arefunctionally linked to one or more regulatory signals which ensuretranscription and translation in plants.

These nucleic acid constructs in which the coding nucleic acid sequenceis functionally linked to one or more regulatory signals which ensuretranscription and translation in plants are also called expressioncassettes below.

The regulatory signals preferably comprise one or more promoters whichensure transcription and translation in plants.

The expression cassettes comprise regulatory signals, i.e. regulatingnucleic acid sequences which control the expression of the codingsequence in the host cell. In a preferred embodiment, an expressioncassette comprises a promoter upstream, i.e. at the 5′ end of the codingsequence, and a polyadenylation signal downstream, i.e. at the 3′ end,and, where appropriate, further regulatory elements which areoperatively linked to the coding sequence, located in between, for atleast one of the genes described above. Operative linkage means thesequential arrangement of promoter, coding sequence, terminator and,where appropriate, further regulatory elements in such a way that eachof the regulatory elements is able to carry out its function as intendedin the expression of the coding sequence.

The preferred nucleic acid constructs, expression cassettes and vectorsfor plants and processes for producing transgenic plants, and thetransgenic plants themselves, are described by way of example below.

The sequences which are preferred for the operative linkage, but are notrestricted thereto, are targeting sequences to ensure the subcellularlocalization in the apoplast, in the vacuole, in plastids, in themitochondrion, in the endoplasmic reticulum (ER), in the cell nucleus,in elaioplasts or other compartments and translation enhancers such asthe 5′ leader sequence from tobacco mosaic virus (Gallie et al., Nucl.Acids Res. 15 (1987), 8693-8711).

A suitable promoter for the expression cassette is in principle anypromoter able to control the expression of foreign genes in plants.

“Constitutive” promoter means promoters which ensure expression innumerous, preferably all, tissues over a relatively wide period duringdevelopment of the plant, preferably at all times during development ofthe plant.

Preferably used is, in particular, a plant promoter or a promoterderived from a plant virus. Particular preference is given to the CaMVpromoter of the ³⁵S transcript of cauliflower mosaic virus (Franck etal. (1980) Cell 21:285-294; Odell et al. (1985) Nature 313:810-812;Shewmaker et al. (1985) Virology 140:281-288; Gardner et al. (1986)Plant Mol Biol 6:221-228), the 19S CaMV promoter (U.S. Pat. No.5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J. 8:2195-2202), thetriose phosphate translocator (TPT) promoter from Arabidopsis thalianaAcc. No. AB006698, base pair 53242 to 55281; the gene starting at bp55282 is anotated as “phosphate/triose phosphate translocator”, or the34S promoter from figwort mosaic virus Acc. No. X16673, base pair 1 to554.

A further suitable constitutive promoter is the pds promoter (Pecker etal. (1992) Proc. Natl. Acad. Sci USA 89: 4962-4966) or the rubisco smallsubunit (SSU) promoter (U.S. Pat. No. 4,962,028), the legumin B promoter(GenBank Acc. No. X03677), the agrobacterium nopaline synthase promoter,the TR dual promoter, the agrobacterium OCS (octopine synthase)promoter, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol29:637-639), the ubiquitin 1 promoter (Christensen et al. (1992) PlantMol Biol 18:675-689; Bruce et al. (1989) Proc Natl Acad Sci USA86:9692-9696), the Smas promoter, the cinnamyl alcohol dehydrogenasepromoter (U.S. Pat. No. 5,633,439), the promoters of the vacuolar ATPasesubunits or the promoter of a proline-rich protein from wheat (WO91/13991), the Pnit promoter (Y07648.L, Hillebrand et al. (1998), Plant.Mol. Biol. 36, 89-99, Hillebrand et al. (1996), Gene, 170, 197-200) andfurther promoters of genes whose constitutive expression in plants isknown to the skilled worker.

The expression cassettes may also comprise a chemically induciblepromoter (review article: Gatz et al. (1997) Annu Rev Plant PhysiotPlant Mol Biol 48:89-108) by which expression of the ketolase gene inthe plant can be controlled at a particular time. Promoters of thistype, such as, for example, the PRP1 promoter (Ward et al. (1993) PlantMol Biol 22:361-366), a salicylic acid-inducible promoter (WO 95/19443),a benzenesulfonamide-inducible promoter (EP 0 388 186), atetracycline-inducible promoter (Gatz et al. (1992) Plant J 2:397404),an abscisic acid-inducible promoter (EP 0 335 528) or an ethanol- orcyclohexanone-inducible promoter (WO 93/21334), can likewise be used.

Promoters which are further preferred are those induced by biotic orabiotic stress, such as, for example, the pathogen-inducible promoter ofthe PRP1 gene (Ward et al. (1993) Plant Mol Biol 22:361-366), theheat-inducible tomato hsp70 or hsp80 promoter (U.S. Pat. No. 5,187,267),the cold-inducible potato alpha-amylase promoter (WO 96/12814), thelight-inducible PPDK promoter or the wound-induced pinII promoter(EP375091).

Pathogen-inducible promoters include those of genes which are induced asa result of pathogen attack, such as, for example, genes of PR proteins,SAR proteins, β-1,3-glucanase, chitinase etc. (for example Redolfi etal. (1983) Neth J Plant Pathol 89:245-254; Uknes, et al. (1992) ThePlant Cell 4:645-656; Van Loon (1985) Plant Mol Viral 4:111-116;Marineau et al. (1987) Plant Mol Biol 9:335-342; Matton et al. (1987)Molecular Plant-Microbe Interactions 2:325-342; Somssich et al. (1986)Proc Natl Acad Sci USA 83:2427-2430; Somssich et al. (1988) Mol GenGenetics 2:93-98; Chen et al. (1996) Plant J 10:955-966; Zhang and Sing(1994) Proc Natl Acad Sci USA 91:2507-2511; Warner, et al. (1993) PlantJ 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968 (1989).

Also included as wound-inducible promoters such as that of the pinIIgene (Ryan (1990) Ann Rev Phytopath 28:425-449; Duan et al. (1996) NatBiotech 14:494-498), of the wun1 and wun2 genes (U.S. Pat. No.5,428,148), of the win1 and win2 genes (Stanford et al. (1989) Mol GenGenet 215:200-208), of the systemin gene (McGurl et al. (1992) Science255:1570-1573), of the WIP1 gene (Rohmeier et al. (1993) Plant Mol Biol22:783-792; Ekelkamp et al. (1993) FEBS Letters 323:73-76), of the MPIgene (Corderok et al. (1994) The Plant J 6(2):141-150) and the like.

Examples of further suitable promoters are fruit ripening-specificpromoters such as, for example, the tomato fruit ripening-specificpromoter (WO 94/21794, EP 409 625). Development-dependent promotersinclude some of the tissue-specific promoters because the formation ofsome tissues naturally depends on development.

Further particularly preferred promoters are those which ensureexpression in tissues or parts of plant in which, for example, thebiosynthesis of ketocarotenoids or precursors thereof takes place.Preferred examples are promoters having specificities for anthers,ovaries, petals, sepals, flowers, leaves, stalks, seeds and roots andcombinations thereof.

Examples of promoters specific for tubers, storage roots or roots arethe patatin promoter class I (B33) or the potato cathepsin D inhibitorpromoter.

Examples of leaf-specific promoters are the promoter of the potatocytosolic FBPase (WO 97/05900), the rubisco (ribulose-1,5-bisphosphatecarboxylase) SSU promoter (small subunit) or the potato ST-LSI promoter(Stockhaus et al., (1989) EMBO J. 8:2445-2451).

Examples of flower-specific promoters are the phytoene synthase promoter(WO 92/16635) or the promoter of the P-rr gene (WO 98/22593), theArabidopsis thaliana AP3 promoter (see example 5), the CHRC promoter(chromoplast-specific carotenoid-associated protein (CHRC) gene promoterfrom Cucumis sativus Acc. No. AF099501, base pair 1 to 1532), the EPSPsynthase promoter (5-enolpyruvylshikimate-3-phosphate synthase genepromoter from Petunia hybrida, Acc. No. M37029, base pair 1 to 1788),the PDS promoter (phytoene desaturase gene promoter from Solanumlycopersicum, Acc. No. U46919, base pair 1 to 2078), the DFR-A promoter(dihydroflavonol 4-reductase gene A promoter from Petunia hybrida, Acc.No. X79723, base pair 32 to 1902) or the FBP1 promoter (floral bindingprotein 1 gene promoter from Petunia hybrida, Acc. No. L10115, base pair52 to 1069).

Examples of anther-specific promoters are the 5126 promoter (U.S. Pat.No. 5,689,049, U.S. Pat. No. 5,689,051), the glob-I promoter or theg-zein promoter.

Examples of seed-specific promoters are the ACPO₅ promoter (acyl carrierprotein gene, WO 9218634), the Arabidopsis AtS1 and AtS3 promoters (WO9920775), the Vicia faba LeB4 promoter (WO 9729200 and U.S. Pat. No.0,640,337,1), the Brassica napus napin promoter (U.S. Pat. No.5,608,152; EP 255378; U.S. Pat. No. 5,420,034), the Vicia faba SBPpromoter (DE 9903432) or the maize End1 and End2 promoters (WO 0011177).

Further promoters suitable for expression in plants are described inRogers et al. (1987) Meth in Enzymol 153:253-277; Schardl et al. (1987)Gene 61:1-11 and Berger et al. (1989) Proc Natl Acad Sci USA86:8402-8406.

Particularly preferred in the process of the invention are constitutive,seed-specific, fruit-specific, flower-specific and, in particular,petal-specific promoters.

The present invention therefore relates in particular to a nucleic acidconstruct comprising functionally linked a flower-specific or, inparticular, a petal-specific promoter and a nucleic acid encoding aketolase comprising the amino acid sequence SEQ. ID. NO. 2 or a sequencewhich is derived from this sequence by substitution, insertion ordeletion of amino acids and which has an identity of at least 42% at theamino acid level with the sequence SEQ. ID. NO. 2.

An expression cassette is preferably produced by fusing a suitablepromoter to a nucleic acid, described above, encoding a ketolase, andpreferably to a nucleic acid which is inserted between promoter andnucleic acid sequence and which codes for a plastid-specific transitpeptide, and to a polyadenylation signal by conventional recombinationand cloning techniques as described, for example in T. Maniatis, E. F.Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and in T. J.Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and inAusubel, F. M. et al., Current Protocols in Molecular Biology, GreenePublishing Assoc. and Wiley-Interscience (1987).

The preferably inserted nucleic acids encoding a plastid transit peptideensure localization in plastids and, in particular, in chromoplasts.

It is also possible to use expression cassettes whose nucleic acidsequence codes for a ketolase fusion protein, where part of the fusionprotein is a transit peptide which controls the translocation of thepolypeptide. Transit peptides which are specific for chromoplasts andwhich are eliminated enzymatically from the ketolase part aftertranslocation of the ketolase into the chromoplasts.

The particularly preferred transit peptide is derived from the Nicotianatabacum plastid transketolase or another transit peptide (e.g. thetransit peptide of the small subunit of rubisco (rbcS) or of theferredoxin NADP⁺ oxidoreductase, as well as theisopentenyl-pyrophosphate isomerase 2) or its functional equivalent.

Particular preference is given to nucleic acid sequences of threecassettes of the plastid transit peptide of the tobacco plastictransketolase in three reading frames as KpnII/BamHI fragments with anATG codon in the NcoI cleavage site: pTP09Kpnl_GGTACCATGGCGTCTTCTTCTTCTCTCACTCTCTCTCAAGCTATCCTCTCTCGTTCTGTCCCTCGCCATGGCTCTGCCTCTTCTTCTCAACTTTCCCCTTCTTCTCTCACTTTTTCCGGCCTTAAATCCAATCCCAATATCACCACCTCCCGCCGCCGTACTCCTTCCTCCGCCGCCGCCGCCGCCGTCGTAAGGTCACCGGCGATTCGTGCCTCAGCTGCAACCGAAACCATAGAGAAAACTGAG ACTGCGGGATCC_BamHlpTP10 KPnl_GGTACCATGGCGTCTTCTTCTTCTCTCACTCTCTCTCAAGCTATCCTCTCTCGTTCTGTCCCTCGCCATGGCTCTGCCTCTTCTTCTCAACTTTCCCCTTCTTCTCTCACTTTTTCCGGCCTTAAATCCAATCCCAATATCACCACCTCCCGCCGCCGTACTCCTTCCTCCGCCGCCGCCGCCGCCGTCGTAAGGTCACCGGCGATTCGTGCCTCAGCTGCAACCGAAACCATAGAGAAAACTGAG ACTGCGCTGGATCC_BamHlpTP11 Kpnl_GGTACCATGGCGTCTTCTTCTTCTCTCACTCTCTCTCAAGCTATCCTCTCTCGTTCTGTCCCTCGCCATGGCTCTGCCTCTTCTTCTCAACTTTCCCCTTCTTCTCTCACTTTTTCCGGCCTTAAATCCAATCCCAATATCACCACCTCCCGCCGCCGTACTCCTTCCTCCGCCGCCGCCGCCGCCGTCGTAAGGTCACCGGCGATTCGTGCCTCAGCTGCAACCGAAACCATAGAGAAAACTGAG ACTGCGGGGATCC_BamHl

Further examples of a plastid transit peptide are the transit peptide ofthe Arabidopsis thaliana plastid isopentenyl-pyrophosphate isomerase 2(IPP-2) and the transit peptide of the small subunit ofribulose-bisphosphate carboxylase (rbcS) from pea (Guerineau, F,Woolston, S, Brooks, L, Mullineaux, P (1988) An expression cassette fortargeting foreign proteins into the chloroplasts. Nucl. Acids Res.16:11380).

The nucleic acids of the invention can be prepared synthetically orobtained naturally or comprise a mixture of synthetic and naturalnucleic acid constituents, and consist of various heterologous genesections from different organisms.

Preference is given, as described above, to synthetic nucleotidesequences with codons preferred by plants. These codons preferred byplants can be identified from codons with the highest protein frequencywhich are expressed in most plant species of interest.

For preparing an expression cassette it is possible to manipulatevarious DNA fragments in order to obtain a nucleotide sequence whichexpediently reads in the correct direction and is equipped with acorrect reading frame. Adaptors or linkers can be attached to thefragments for connecting the DNA fragments to one another.

It is possible and expedient for the promoter and terminator regions tobe provided in the direction of transcription with a linker orpolylinker which contains one or more restriction sites for insertingthis sequence. As a rule, the linker has 1 to 10, usually 1 to 8,preferably 2 to 6, restriction sites. The linker generally has a size ofless than 100 bp, frequently less than 60 bp, but at least 5 bp, insidethe regulatory regions. The promoter may be both native or homologousand foreign or heterologous to the host plant. The expression cassettepreferably comprises in the 5′-3′ direction of transcription thepromoter, a coding nucleic acid sequence or a nucleic acid construct anda region for termination of transcription. Various termination regionsare interchangeable as desired.

Examples of a terminator are the 35S terminator (Guerineau et al. (1988)Nuci Acids Res. 16: 11380), the nos terminator (Depicker A, Stachel S,Dhaese P, Zambryski P, Goodman H M. Nopaline synthase: transcriptmapping and DNA sequence. J Mol Appl Genet. 1982; 1(6):561-73) or theocs terminator (Gielen, J, de Beuckeleer, M, Seurinck, J, Debroek, H, deGreve, H, Lemmers, M, van Montagu, M, Schell, J (1984) The completesequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5.EMBO J. 3: 835-846).

It is furthermore possible to employ manipulations which provideappropriate restriction cleavage sites or delete the redundant DNA orrestriction cleavage sites. It is possible in relation to insertions,deletions or substitutions, such as, for example, transitions andtransversions, to use in vitro mutagenesis, primer repair, restrictionor ligation.

It is possible with suitable manipulations, such as, for example,restriction, chewing back or filling in of overhangs for blunt ends, toprovide complementary ends of the fragments for ligation.

Preferred polyadenylation signals are plant polyadenylation signals,preferably those which essentially correspond to T-DNA polyadenylationsignals from Agrobacterium tumefaciens, especially of gene 3 of theT-DNA (octopine synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBOJ. 3 (1984), 835 ff) or functional equivalents.

The transfer of foreign genes into the genome of a plant is referred toas transformation.

It is possible to use for this purpose methods known per se for thetransformation and regeneration of plants from plant tissues or plantcells for transient or stable transformation.

Suitable methods for transforming plants are protoplast transformationby polyethylene glycol-induced DNA uptake, the biolistic method usingthe gene gun—called the particle bombardment method—electroporation,incubation of dry embryos in DNA-containing solution, microinjection andgene transfer mediated by Agrobacterium described above. Said processesare described, for example, in B. Jenes et al., Techniques for GeneTransfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization,edited by S. D. Kung and R. Wu, Academic Press (1993), 128-143 and inPotrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991),205-225.

The construct to be expressed is preferably cloned into a vector whichis suitable for transforming Agrobacterium tumefaciens, for examplepBin19 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711) or particularlypreferably, pSUN2, pSUN3, pSUN4 or pSUN5 (WO 02/00900).

Agrobacteria transformed with an expression plasmid can be used in aknown manner for transforming plants, e.g. bathing wounded leaves orpieces of leaf in a solution of agrobacteria and subsequentlycultivating in suitable media.

For the preferred production of genetically modified plants, alsoreferred to as transgenic plants hereinafter, the fused expressioncassette which expresses a ketolase is cloned into a vector, for examplepBin19 or, in particular, pSUN5 and pSUN3, which is suitable for beingtransformed into Agrobacterium tumefaciens. Agrobacteria transformedwith such a vector can then be used in a known manner for transformingplants, in particular crop plants, by bathing wounded leaves or piecesof leaf in a solution of agrobacteria and subsequently cultivating insuitable media.

The transformation of plants by agrobacteria is disclosed inter alia inF. F. White, Vectors for Gene Transfer in Higher Plants; in TransgenicPlants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R.Wu, Academic Press, 1993, pages 15-38. Transgenic plants which comprisea gene, integrated into the expression cassette for expression of anucleic acid encoding a ketolase can be regenerated in a known mannerfrom the transformed cells of the wounded leaves or pieces of leaf.

To transform a host cell with a nucleic acid coding for a ketolase, anexpression cassette is incorporated and inserted into a recombinantvector whose vector DNA comprises additional functional regulatorysignals, for example sequences for replication or integration. Suitablevectors are described inter alia in “Methods in Plant Molecular Biologyand Biotechnology” (CRC Press), chapter 6/7, pages 71-119 (1993).

Using the recombination and cloning techniques quoted above, theexpression cassettes can be cloned into suitable vectors which makereplication thereof possible for example in E. coli. Suitable cloningvectors are, inter alia, pJIT117 (Guerineau et al. (1988) Nucl. AcidsRes. 16:11380), pBR322, pUC series, M13 mp series and pACYC184. Binaryvectors which are able to replicate both in E. coli and in agrobacteriaare particularly suitable.

The production of the genetically modified microorganisms of theinvention is described in more detail below:

The nucleic acids described above, encoding a ketolase or hydroxylase orβ-cyclase, are preferably incorporated into expression constructscomprising, under the genetic control of regulatory nucleic acidsequences, a nucleic acid sequence coding for an enzyme of theinvention; and vectors comprising at least one of these expressionconstructs.

Such constructs of the invention preferably include a promoter upstream,i.e. at the 5′ end of the particular coding sequence, and a terminatorsequence downstream, i.e. at the 3′ end, and, where appropriate, furthercustomary regulatory elements which are in each case operatively linkedto the coding sequence. Operative linkage means the sequentialarrangement of promoter, coding sequence, terminator and, whereappropriate, further regulatory elements in such a way that each of theregulatory elements is able to carry out its function as intended in theexpression of the coding sequence.

Examples of operatively linkable sequences are targeting sequences andtranslation enhancers, polyadenylation signals and the lilke. Furtherregulatory elements include selectable markers, amplification signals,origins of replication and the like.

In addition to the artificial regulatory sequences it is possible forthe natural regulatory sequence still to be present in front of theactual structural gene. This natural regulation can be switched offwhere appropriate, and the expression of the genes increased or reduced,by genetic modification. The gene construct may, however, also have asimpler structure, that is to say no additional regulatory signals areinserted in front of the structural gene, and the natural promoter withits regulation is not deleted. Instead, the natural regulatory sequenceis mutated so that regulation no longer takes place, and gene expressionis increased or reduced. The nucleic acid sequences may be present inone or more copies in the gene construct.

Examples of promoters which can be used are: cos, tac, trp, tet,trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6,lambda-PR or lambda-PL promoter, which are advantageously used inGram-negative bacteria; and the Gram-positive promoters amy and SPO2 orthe yeast promoters ADC1, MFα, AC, P-60, CYC1, GAPDH. The use ofinducible promoters is particularly preferred, such as, for example,light- and, in particular, temperature-inducible promoters such as theP_(r)P_(l) promoter.

It is possible in principle for all natural promoters with theirregulatory sequences to be used. In addition, it is also possibleadvantageously to use synthetic promoters.

Said regulatory sequences are intended to make specific expression ofthe nucleic acid sequences and protein expression possible. This maymean, for example, depending on the host organism, that the gene isexpressed or overexpressed only after induction or that it isimmediately expressed and/or overexpressed.

The regulatory sequences or factors may moreover preferably influencepositively, and thus increase or reduce, expression. Thus, enhancementof the regulatory elements can take place advantageously at the level oftranscription by using strong transcription signals such as promotersand/or enhancers. However, it is also possible to enhance translationby, for example, improving the stability of the mRNA.

An expression cassette is produced by fusing a suitable promoter to theabove described nucleic acid sequence which encodes a ketolase,β-hydroxylase or β-cyclase and to a terminator signal or polyadenylationsignal. Conventional techniques of recombination and cloning are usedfor this purpose, as described, for example, in T. Maniatis, E. F.Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and in T. J.Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and inAusubel, F. M. et al., Current Protocols in Molecular Biology, GreenePublishing Assoc. and Wiley Interscience (1987).

For expression in a suitable host organism, the recombinant nucleic acidconstruct or gene construct is advantageously inserted into ahost-specific vector, which makes optimal expression of the genes in thehost possible. Vectors are well known to the skilled worker and can befound, for example, in “Cloning Vectors” (Pouwels P. H. et al., eds,Elsevier, Amsterdam-New York-Oxford, 1985). Vectors also mean not onlyplasmids but also all other vectors known to the skilled worker, suchas, for example, phages, viruses, such as SV40, CMV, baculovirus andadenovirus, transposons, IS elements, phasmids, cosmids, and linear orcircular DNA. These vectors may undergo autonomous replication in thehost organism or chromosomal replication.

Examples of suitable expression vectors which may be mentioned are:

Conventional fusion expression vectors such as pGEX (Pharmacia BiotechInc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT 5 (Pharmacia, Piscataway,N.J.), with which respectively glutathione S-transferase (GST), maltoseE-binding protein and protein A are fused to the recombinant targetprotein.

Non-fusion protein expression vectors such as pTrc (Amann et al., (1988)Gene 69:301-315) and pET 11d (Studier et al. Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)60-89).

Yeast expression vector for expression in the yeast S. cerevisiae, suchas pYepSec1 (Baldari et al., (1987) Embo J. 6:229-234), pMFα (Kurjan andHerskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene54:113-123) and pYES2 (Invitrogen Corporation, San Diego, Calif.).

Vectors and methods for constructing vectors suitable for the use inother fungi such as filamentous fungi comprise those which are describedin detail in: van den Hondel, C. A. M. J. J. & Punt, P. J. (1991) “Genetransfer systems and vector development for filamentous fungi, in:Applied Molecular Genetics of Fungi, J. F. Peberdy et al., eds, pp.1-28, Cambridge University Press: Cambridge.

Baculovirus vectors which are available for expression of proteins incultured insect cells (for example Sf9 cells) comprise the pAc series(Smith et al., (1983) Mol. Cell Biol. 3:2156-2165) and pVL series(Lucklow and Summers (1989) Virology 170:31-39).

Further suitable expression systems for prokaryotic and eukaryotic cellsare described in chapters 16 and 17 of Sambrook, J., Fritsch, E. F. andManiatis, T., Molecular cloning: A Laboratory Manual, 2nd edition, ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

The expression constructs or vectors of the invention can be used toproduce genetically modified microorganisms which are transformed, forexample, with at least one vector of the invention.

The recombinant constructs of the invention described above areadvantageously introduced and expressed in a suitable host system.Cloning and transfection methods familiar to the skilled worker, suchas, for example, coprecipitation, protoplast fusion, electroporation,retroviral transfection and the like, are preferably used to bring aboutexpression of said nucleic acids in the particular expression system.Suitable systems are described, for example, in Current Protocols inMolecular Biology, F. Ausubel et al., eds, Wiley Interscience, New York1997.

Successfully transformed organisms can be selected through marker geneswhich are likewise present in the vector or in the expression cassette.Examples of such marker genes are genes for antibiotic resistance andfor enzymes which catalyze a color-forming reaction which causesstaining of the transformed cell. These can then be selected byautomatic cell sorting.

Microorganisms which have been successfully transformed with a vectorand harbor an appropriate antibiotic resistance gene (for example G418or hygromycin) can be selected by appropriate antibiotic-containingmedia or nutrient media. Marker proteins present on the surface of thecell can be used for selection by means of affinity chromatography.

The combination of the host organisms and the vectors appropriate forthe organisms, such as plasmids, viruses or phages, such as, forexample, plasmids with the RNA polymerase/promoter system, phages 8 orother temperate phages or transposons and/or other advantageousregulatory sequences forms an expression system.

The invention further relates to a process for producing geneticallymodified organisms, which comprises introducing a nucleic acid constructcomprising functionally linked a promoter and nucleic acids encoding aketolase comprising the amino acid sequence SEQ. ID. NO. 2 or a sequencewhich is derived from this sequence by substitution, insertion ordeletion of amino acids and which has an identity of at least 42% at theamino acid level with the sequence SEQ. ID. NO. 2, and, whereappropriate, a terminator into the genome of the starting organism orextrachromosomally into the starting organism.

The invention further relates to the genetically modified organismswhere the genetic modification

-   A in the case where the wild-type organism already has a ketolase    activity, increases the activity of a ketolase compared with the    wild type and-   B in the case where the wild-type organism has no ketolase activity,    causes the activity of a ketolase compared with the wild type,    and the ketolase activity which has been increased as in A or caused    as in B is caused by a ketolase comprising the amino acid sequence    SEQ. ID. NO. 2 or a sequence which is derived from this sequence by    substitution, insertion or deletion of amino acids and which has an    identity of at least 42% at the amino acid level with the sequence    SEQ. ID. NO. 2.

As stated above, the increasing or causing of the ketolase activity isbrought about by an increasing or causing of the gene expression of anucleic acid encoding a ketolase comprising the amino acid sequence SEQ.ID. NO. 2 or a sequence which is derived from this sequence bysubstitution, insertion or deletion of amino acids and which has anidentity of at least 42% at the amino acid level with the sequence SEQ.ID. NO. 2, compared with the wild type.

In a further preferred embodiment, as stated above, the increasing orcausing of the gene expression of a nucleic acid encoding a ketolasetakes place by introducing nucleic acids encoding a ketolase into theplants and thus preferably by overexpression or transgenic expression ofnucleic acids encoding a ketolase comprising the amino acid sequenceSEQ. ID. NO. 2 or a sequence which is derived from this sequence bysubstitution, insertion or deletion of amino acids and which has anidentity of at least 42% at the amino acid level with the sequence SEQ.ID. NO. 2.

The invention further relates to a genetically modified organismcomprising at least one transgenic nucleic acid encoding a ketolasecomprising the amino acid sequence SEQ. ID. NO. 2 or a sequence which isderived from this sequence by substitution, insertion or deletion ofamino acids and which has an identity of at least 42% at the amino acidlevel with the sequence SEQ. ID. NO. 2. This is the case when thestarting organism has no ketolase or an endogenous ketolase, and atransgenic ketolase is overexpressed.

The invention further relates to a genetically modified organismcomprising at least two endogenous nucleic acids encoding a ketolasecomprising the amino acid sequence SEQ. ID. NO. 2 or a sequence which isderived from this sequence by substitution, insertion or deletion ofamino acids and which has an identity of at least 42% at the amino acidlevel with the sequence SEQ. ID. NO. 2. This is the case when thestarting organism has an endogenous ketolase, and the endogenousketolase is overexpressed.

Particularly preferred genetically modified organisms have, as mentionedabove, additionally an increased hydroxylase activity and/or β-cyclaseactivity compared with a wild-type organism. Further preferredembodiments are described above in the process of the invention.

Organisms preferably mean according to the invention organisms which areable as wild-type or starting organisms naturally or through geneticcomplementation and/or reregulation of metabolic pathways to producecarotenoids, in particular β-carotene and/or zeaxanthin and/orneoxanthin and/or violaxanthin and/or luteine.

Further preferred organisms already have as wild-type or startingorganisms a hydroxylase activity and are thus able as wild-type orstarting organisms to produce zeaxanthin.

Preferred organisms are plants or microorganisms such as, for example,bacteria, yeasts, algae or fungi.

Bacteria which can be used are both bacteria which are able, because ofthe introduction of genes of carotenoid biosynthesis of acarotenoid-producing organism, to synthesize xanthophylls, such as, forexample, bacteria of the genus Escherichia, which comprise for examplecrt genes from Erwinia, and bacteria which are intrinsically able tosynthesize xanthophylls, such as, for example, bacteria of the genusErwinia, Agrobacterium, Flavobacterium, Alcaligenes, Paracoccus, Nostocor cyanobacteria of the genus Synechocystis.

Preferred bacteria are Escherichia coli, Erwinia herbicola, Erwiniauredovora, Agrobacterium aurantiacum, Alcaligenes sp. PC-1,Flavobactenum sp. strain R1534, the cyanobacterium Synechocystis sp.PCC6803, Paracoccus marcusii or Paracoccus carotinifaciens.

Preferred yeasts are Candida, Saccharomyces, Hansenula, Pichia orPhaffia. Particularly preferred yeasts are Xanthophyllomyces dendrorhousor Phaffia rhodozyma.

Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora,Blakeslea, Phycomyces, Fusarium or other fungi described in Indian Chem.Engr. Section B. Vol. 37, No. 1, 2 (1995) on page 15, table 6.

Preferred algae are green algae such as, for example, algae of the genusHaematococcus, Phaedactylum tricomatum, Volvox or Dunaliella.Particularly preferred algae are Haematococcus pluvialis or Dunaliellabardawil.

Further microorganisms which can be used and the production thereof forcarrying out the process of the invention are disclosed for example inDE-A-199 16 140, which is incorporated herein by reference.

Particularly preferred plants are plants selected from the familiesRanunculaceae, Berberidaceae, Papaveraceae, Cannabaceae, Rosaceae,Fabaceae, Linaceae, Vitaceae, Brassicaceae, Cucurbitaceae, Primulaceae,Caryophyllaceae, Amaranthaceae, Gentianaceae, Geraniaceae,Caprifoliaceae, Oleaceae, Tropaeolaceae, Solanaceae, Scrophulariaceae,Asteraceae, Liliaceae, Amaryllidaceae, Poaceae, Orchidaceae, Malvaceae,llliaceae or Lamiaceae.

Very particularly preferred plants are selected from the group of plantgenera Marigold, Tagetes errecta, Tagetes patula, Acacia, Aconitum,Adonis, Amica, Aquilegia, Aster, Astragalus, Bignonia, Calendula,Caftha, Campanula, Canna, Centaurea, Cheiranthus, Chrysanthemum, Citrus,Crepis, Crocus, Curcurbita, Cytisus, Delonia, Delphinium, Dianthus,Dimorphotheca, Doronicum, Eschscholtzia, Forsythia, Fremontia, Gazania,Gelsemium, Genista, Gentiana, Geranium, Gerbera, Geum, Grevillea,Helenium, Helianthus, Hepatica, Heracleum, Hibiscus, Heliopsis,Hypencum, Hypochoeris, Impatiens, Iris, Jacaranda, Kerria, Labumum,Lathyrus, Leontodon, Lilium, Linum, Lotus, Lycopersicon, Lysimachia,Maratia, Medicago, Mimulus, Narcissus, Oenothera, Osmanthus, Petunia,Photinia, Physalis, Phyteuma, Potentilla, Pyracantha, Ranunculus,Rhododendron, Rosa, Rudbeckia, Senecio, Silene, Silphium, Sinapsis,Sorbus, Spartium, Tecoma, Torenia, Tragopogon, Trollius, Tropaeolum,Tulipa, Tussilago, Ulex, Viola or Zinnia, particularly preferablyselected from the group of plant genera Marigold, Tagetes erecta,Tagetes patula, Lycopersicon, Rosa, Calendula, Physalis, Medicago,Helianthus, Chrysanthemum, Aster, Tulipa, Narcissus, Petunia, Geranium,Tropaeolum or Adonis.

Very particularly preferred genetically modified plants are selectedfrom the plant genera Marigold, Tagetes erecta, Tagetes patula, Adonis,Lycopersicon, Rosa, Calendula, Physalis, Medicago, Helianthus,Chrysanthemum, Aster, Tulipa, Narcissus, Petunia, Geranium orTropaeolum, with the genetically modified plant comprising at least onetransgenic nucleic acid encoding a ketolase.

The present invention further relates to the transgenic plants, theirpropagation material, and their plant cells, tissues or parts,especially their fruit, seeds, flowers and petals.

The genetically modified plants can, as described above, be used forpreparing ketocarotenoids, especially astaxanthin.

Genetically modified organisms of the invention which can be consumed byhumans and animals, especially plants or parts of plants, such as, inparticular, petals with an increased content of ketocarotenoids,especially astaxanthin, can also be used directly or after processingknown per se as human or animal foods or as animal and human foodsupplements.

The genetically modified organisms can also be used for producingketocarotenoid-containing extracts of the organisms and/or for producinganimal and human food supplements.

The genetically modified organisms have an increased content ofketocarotenoids compared with the wild type.

An increased content of ketocarotenoids usually means an increased totalketocarotenoid content.

However, an increased content of ketocarotenoid also means in particularan altered content of the preferred ketocarotenoids without the need forthe total carotenoid content necessarily to be increased.

In a particularly preferred embodiment, the genetically modified plantsof the invention have an increased astaxanthin content compared with thewild type.

An increased content means in this case also a caused content ofketocarotenoids such as astaxanthin.

The invention further relates to the novel ketolases and to the novelnucleic acids which encode the latter.

The invention relates in particular to ketolases comprising the aminoacid sequence SEQ. ID. NO. 2 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhas an identity of at least 70%, preferably at least 75%, particularlypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 95% at the amino acid level withthe sequence SEQ. ID. NO. 2, with the proviso that the amino acidsequence SEQ. ID NO. 2 is not present. The sequence SEQ ID NO: 2 is, asmentioned above, annotated as putative protein in databases.

The invention further relates to ketolases comprising the amino acidsequence SEQ. ID. NO. 4 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhas an identity of at least 70% at the amino acid level with thesequence SEQ. ID. NO. 4. The sequence SEQ ID NO: 4 is, as mentionedabove, not annotated in databases.

The invention further relates to nucleic acids encoding a proteindescribed above, with the proviso that the nucleic acid does notcomprise the sequences SEQ ID NO: 1 or 3.

It has surprisingly been found that a protein comprising the amino acidsequence SEQ. ID. NO. 2 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhas an identity of at least 70%, preferably at least 75%, particularlypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 95%, at the amino acid level withthe sequence SEQ. ID. NO. 2 and has the property of a ketolase, has aproperty as ketolase.

The invention therefore also relates to the use of a protein comprisingthe amino acid sequence SEQ. ID. NO. 2 or a sequence which is derivedfrom this sequence by substitution, insertion or deletion of amino acidsand which has an identity of at least 70%, preferably at least 75%,particularly preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 95%, at the amino acidlevel with the sequence SEQ. ID. NO. 2, and has the property of aketolase, as ketolase.

It has also surprisingly been found that a protein comprising the aminoacid sequence SEQ. ID. NO. 4 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhas an identity of at least 65%, preferably at least 70%, preferably atleast 75%, particularly preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, more preferably at least 95%,at the amino acid level with the sequence SEQ. ID. NO. 4, and has theproperty of a ketolase, has a property as ketolase.

The invention therefore also relates to the use of a protein comprisingthe amino acid sequence SEQ. ID. NO. 4 or a sequence which is derivedfrom this sequence by substitution, insertion or deletion of amino acidsand which has an identity of at least 65%, preferably at least 70%,preferably at least 75%, particularly preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 95%, at the amino acid level with the sequence SEQ. ID. NO. 4,and has the property of a ketolase, as ketolase.

Compared with prior art processes, the process of the invention affordsa larger quantity of ketocarotenoids, especially astaxanthin.

The invention is now explained by the following examples, but is notrestricted thereto:

EXAMPLE 1

Amplification of cDNA, which encodes a complete primary sequence of theketolases from Nostocpunctiforme PCC73102 ORF 38, contig 501 (SEQ IDNO: 1) and ORF 148, contig 502 (SEQ ID NO: 3)

Cells of Nostoc punctiforme were disrupted with lysozyme (2 mg/ml) andthe genomic DNA was isolated with the aid of the GenElute Plant genomicDNA kit (Sigma) in accordance with the manufacturer's information.

This was followed by amplification of ORF148 (762 bp) from the genomicDNA of Nostoc punctiforme with the aid of the primers 148-Start (SEQ IDNO: 9; 5′ ATG ATC CAG TTA GM CAA CCA C-3′) and 148-End (SEQ ID NO: 10;5′CTA TTT TGC TTT GTA AAT TTC TGG-3′) at an annealing temperature of 60°C. over 30 cycles.

ORF38 (789 bp) was amplified using the primers 38-Start (SEQ ID NO: 11;5′ ATG AAT TTT TGT GAT MA CCA GTT AG-3′) and 38-End (SEQ ID NO: 12; 5′ACG MT TGG TTA CTG MT TGT TG-3′).

The PCR fragments were subcloned into the Xcml-cut vector pMON 38201(Borokov, A. Y. and Rivkin, M. I. (1997) Xcml containing vector fordirect cloning of pcr products. BioTech. 22, 812-814).

Positive clones were selected by carrying out a blue-white screeningafter transformation of the ligation products into XL1 blue MRF1′. Theisolated plasmid DNA was cut with HindIII in order to check whether thePCR amplicon was cloned into the T overhang vector. Sequencing of theselected clones showed that the orientation of ORF148 in pMONT-148, andof ORF38 in pMONT-38, is contrary to the vectorial reading direction. Itwas possible to cut out the insert with HindIII because the T overhangvector possesses not only the HindIII cleavage site in the polylinkerbut also a second one produced on insertion of the polylinker.

EXAMPLE 2

Preparation of expression vectors for expression of the Nostocpunctiforme PCC73102 ketolases ORF148 and ORF38 in host organisms.

After restriction digestion of pMONT148 and pMONT38 with HindIII, theresulting DNA inserts were cloned into a pPQE32 vector (Qiagen, Hilden;modified as described in Verdoes, J., Krubasik, P., Sandmann, G. & vanOoyen, M. (1999) Isolation and functional characterisation of a noveltype of carotenoid biosynthetic gene from Xanthophyllomyces dendrorhous.Molec. Gen. Genet. 262, 453-461 which had likewise been digested withHindIII and dephosphorylated.

The clones obtained after transformation into XL1MRF1′ were examined bymeans of a check PCR using primer QEF (5′CCC TTT CCT CTT CTC-3′) and148-end or 38-end. The sequencings of the corresponding clones showedthat ORF148 and ORF38 were cloned in frame ito the pPQE32 vector. Theplasmids obtained in this way are depicted in FIGS. 2B and 2C. FIG. 2shows the construction of pPQE32-ORF 148 (B.) and pPQE32-ORF 38 (C.)starting from pPQE32 (A.).

EXAMPLE 3

Expression of the Nostoc punctiforme PCC73102 ketolases ORF148 and ORF38in β-carotene and zeaxanthin producing E. coli strains and analysis ofthe carotenoid profile

3.1. Expression of the Nostoc punctiforme PCC73102 ketolases ORF148 andORF38 in β-carotene producing E. coli strains

For functional characterization of the gene products formed by ORF148and ORF38, the constructs pPQE32-148 and pPQE32-38 were transformed intothe β-carotene producing E. coli transformant JM101/pACCAR16ΔcrtX(Misawa, N., Satomi, Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito,T. Ohtani, T. & Miki, W. (1995) Structure and functional analysis of amarine bacterial carotenoid biosynthesis gene cluster and astaxanthinbiosynthetic pathway proposed at the gene level. J. Bacteriol. 22,6575-6584).

The transformants were cultured in 50 ml cultures with LB medium at 28°C. in the dark for 16 to 48 hours. The carotenoids were extracted withmethanol, and the extracts obtained by shaking with 50% ether/petroleumether were fractionated by HPLC (column HypurityC18, mobile phase:acetonitrile/methanol/2-propanol 85:10:5, temperature 32° C.). Thespectra were recorded on-line by means of a diode array detector, andthe carotenoids were identified on the basis of their absorption maximaand by comparison with standards.

As shown in FIG. 3A for pPQE32-38 and 3B for pPQE32-148, it was possibleto detect, besides an initial substrate β-carotene, in both extracts theketocarotenoids echinenone and canthaxanthin (in controls withoutpPQE32-38 or pPQE32-148, only β-carotene but no ketocarotenoids was tobe found).

The proportion of canthaxarithin (diketo compound) produced in the totalcarotenoid content was 81% on complementation with pPQE32-148 and 40% oncomplementation with pPQE32-38. The proportion of echinenone (monoketocompound) was about 4% with both complementations.

3.2. Expression of the Nostoc punctiforme PCC73102 ketolases ORF148 andORF38 in zeaxanthin producing E. coli strains

In order to investigate how far the ketolases encoded by ORF148 andORF38 are able to synthesize the ketocarotenoid astaxanthin, pPQE32-38(FIG. 3C) and pPQE32-148 (FIG. 3D) were transformed into the zeaxanthinproducing E. coli transformants JM101/pACCAR25ΔcrtX (Misawa, N., Satomi,Y., Kondo, K., Yokoyama, A., Kajiwara, S., Saito, T. Ohtani, T. & Miki,W. (1995) Structure and functional analysis of a marine bacterialcarotenoid biosynthesis gene cluster and astaxanthin biosyntheticpathway proposed at the gene level. J. Bacteriol. 22, 6575-6584).

The culturing of the transformants, the carotenoid extraction and theHPLC separation took place as described above in 3.1. Whereas only theinitial substrates zeaxanthin and β-carotene, respectively 85 and 5% ofthe total carotenoid content, were detectable in the extract obtainedfrom complementation with pPQE32-38, chiefly the ketocarotenoidsechinenone, canthaxanthin and astaxanthin were detectable oncomplementation with pPQE32-148. The proportion of astaxanthin in thetotal carotenoid content was 50%. The intermediates of astaxanthinsynthesis, echinenone and canthaxanthin, represent respectively 12% and8% of the total carotenoid. The proportion of β-carotene is about 30%.

FIG. 3 shows the HPLC separation of the carotenoids from complementationin E. coli with a β-carotene background cotransformed with pPQE32-38 (A)or pPQE32-148 (B) and in E. coli with a zeaxanthin backgroundcotransformed with pPQE32-38 (C) or pPQE32-148 (D).

The stated carotenoids were identified by cochromatography withcomparison substances and via their spectra as:

-   1 Canthaxanthin,-   2 Echinenone,-   3 β-Carotene,-   4 Zeaxanthin,-   5 astaxanthin,-   6 β-Cryptoxanthin,-   7 Neurosporin.

1′, 3′, 4′ and 5′ designate the corresponding cis isomers.

1. A process for preparing ketocarotenoids by cultivating geneticallymodified organisms which, compared with the wild type, have a modifiedketolase activity, and the modified ketolase activity is caused by aketolase comprising the amino acid sequence SEQ ID NO: 2 or a sequencewhich is derived from this sequence by substitution, insertion ordeletion of amino acids and which has an identity of at least 42% at theamino acid level with the sequence SEQ ID NO:
 2. 2. The process asclaimed in claim 1, wherein organisms which, as wild type, already havea ketolase activity, and the genetic modification brings about anincrease in the ketolase activity compared with the wild type, are used.3. The process as claimed in claim 1, wherein the ketolase activity isincreased by increasing the gene expression of a nucleic acid encoding aketolase comprising the amino acid sequence SEQ ID NO: 2 or a sequencewhich is derived from this sequence by substitution, insertion ordeletion of amino acids and which has an identity of at least 42% at theamino acid level with the sequence SEQ ID NO: 2, compared with the wildtype.
 4. The process as claimed in claim 3, wherein the gene expressionis increased by introducing nucleic acids which encode ketolasescomprising the amino acid sequence SEQ ID NO: 2 or a sequence which isderived from this sequence by substitution, insertion or deletion ofamino acids and which has an identity of at least 42% at the amino acidlevel with the sequence SEQ ID NO: 2, compared with the wild type, intothe organism.
 5. The process as claimed in claim 1, wherein organismswhich, as wild type, have no ketolase activity are used, and the geneticmodification causes a ketolase activity compared with the wild type. 6.The process as claimed in claim 5, wherein genetically modifiedorganisms which transgenically express a ketolase comprising the aminoacid sequence SEQ ID NO: 2 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhas an identity of at least 42% at the amino acid level with thesequence SEQ ID NO: 2, are used.
 7. The process as claimed in claim 5,wherein the gene expression is caused by introducing nucleic acids whichencode ketolases comprising the amino acid sequence SEQ ID NO: 2 or asequence which is derived from this sequence by substitution, insertionor deletion of amino acids and which has an identity of at least 42% atthe amino acid level with the sequence SEQ ID NO: 2, into the organism8. The process as claimed in claim 5, wherein nucleic acids comprisingthe sequence SEQ ID NO:1 are introduced.
 9. The process as claimed inclaim 1, wherein the organisms additionally have an increased activity,compared with the wild type, of at least one of the activities selectedfrom the group consisting of hydroxylase activity and β-cyclaseactivity.
 10. The process as claimed in claim 9, wherein the geneexpression of at least one nucleic acid selected from the groupconsisting of nucleic acids encoding a hydroxylase, and nucleic acidsencoding a β-cyclase, is increased compared with the wild type for theadditional increase in at least one of the activities.
 11. The processas claimed in claim 10, wherein the gene expression is increased byintroducing at least one nucleic acid selected from the group consistingof nucleic acids encoding a hydroxylase and nucleic acids encoding aβ-cyclase into the organism.
 12. The process as claimed in claim 11,wherein nucleic acids which encode a hydroxylase comprising the aminoacid sequence SEQ ID NO: 6 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhas an identity of at least 20% at the amino acid level with thesequence SEQ ID NO: 6 are introduced as nucleic acid encoding ahydroxylase.
 13. The process as claimed in claim 12, wherein nucleicacids comprising the sequence SEQ ID NO: 5 are introduced.
 14. Theprocess as claimed in claim 11, wherein nucleic acids which encode aβ-cyclase comprising the amino acid sequence SEQ ID NO: 8 or a sequencewhich is derived from this sequence by substitution, insertion ordeletion of amino acids and which has an identity of at least 20% at theamino acid level with the sequence SEQ ID NO: 8 are introduced asnucleic acid encoding a β-cyclase.
 15. The process as claimed in claim14, wherein nucleic acids comprising the sequence SEQ ID NO: 7 areintroduced.
 16. The process as claimed in claim 1, wherein thegenetically modified organisms are harvested after cultivation, andsubsequently the ketocarotenoids are isolated from the organisms. 17.The process as claimed in claim 1, wherein an organism which is able asstarting organism naturally or through genetic complementation orreregulation of metabolic pathways to produce carotenoids is used asorganism.
 18. The process as claimed in claim 1, wherein microorganismsor plants are used as organisms.
 19. The process as claimed in claim 18,wherein bacteria, yeasts, algae or fungi are used as microorganisms. 20.The process as claimed in claim 19, wherein the microorganisms areselected from the group consisting of Escherichia, Erwinia,Agrobacterium, Flavobacterium, Alcaligenes, Paracoccus, Nostoc,cyanobacteria of the genus Synechocystis, Candida, Saccharomyces,Hansenula, Phaffia, Pichia, Aspergillus, Trichoderma, Ashbya,Neurospora, Blakeslea, Phycomyces, Fusarium, Haematococcus, Phaedactylumtricornatum, Volvox and Dunaliella.
 21. The process as claimed in claim18, wherein plants are used as organism.
 22. The process as claimed inclaim 21, wherein a plant selected from the families consisting ofRanunculaceae, Berberidaceae, Papaveraceae, Cannabaceae, Rosaceae,Fabaceae, Linaceae, Vitaceae, Brassiceae, Cucurbitaceae, Primulaceae,Caryophyllaceae, Amaranthaceae, Gentianaceae, Geraniaceae,Caprifoliaceae, Oleaceae, Tropaeolaceae, Solanaceae, Scrophulariaceae,Asteraceae, Liliaceae, Amaryllidaceae, Poaceae, Orchidaceae, Malvaceae,Illiaceae and Lamiaceae is used as plant.
 23. The process as claimed inclaim 22, wherein a plant selected from the plant genera consisting ofMarigold, Tagetes erecta, Tagetes patula, Acacia, Aconitum, Adonis,Arnica, Aquilegia, Aster, Astragalus, Bignonia, Calendula, Caltha,Campanula, Canna, Centaurea, Cheiranthus, Chrysanthemum, Citrus, Crepis,Crocus, Curcurbita, Cytisus, Delonia, Delphinium, Dianthus,Dimorphotheca, Doronicum, Eschscholtzia, Forsythia, Fremontia, Gazania,Gelsemium, Genista, Gentiana, Geranium, Gerbera, Geum, Grevillea,Helenium, Helianthus, Hepatica, Heracleum, Hibiscus, Heliopsis,Hypericum, Hypochoeris, Impatiens, Iris, Jacaranda, Kerria, Laburnum,Lathyrus, Leontodon, Lilium, Linum, Lotus, Lycopersicon, Lysimachia,Maratia, Medicago, Mimulus, Narcissus, Oenothera, Osmanthus, Petunia,Photinia, Physalis, Phyteuma, Potentilla, Pyracantha, Ranunculus,Rhododendron, Rosa, Rudbeckia, Senecio, Silene, Silphium, Sinapsis,Sorbus, Spartium, Tecoma, Torenia, Tragopogon, Trollius, Tropaeolum,Tulipa, Tussilago, Ulex, Viola and Zinnia is used as plant.
 24. Theprocess as claimed in claim 1, wherein the ketocarotenoids are selectedfrom the group consisting of astaxanthin, canthaxanthin, echinenone,3-hydroxyechinenone, 3′-hydroxyechinenone, adonirubin and adonixanthin.25. A genetically modified organism where the genetic modification A, inthe case where the wild-type organism already has a ketolase activity,increases the activity of a ketolase compared with the wild type and B,in the case where the wild-type organism has no ketolase activity,causes the activity of a ketolase compared with the wild type, and theketolase activity which has been increased as in A or caused as in B iscaused by a ketolase comprising the amino acid sequence SEQ ID NO: 2 ora sequence which is derived from this sequence by substitution,insertion or deletion of amino acids and which has an identity of atleast 42% at the amino acid level with the sequence SEQ ID NO:
 2. 26.The genetically modified organism as claimed in claim 25, wherein theincreasing or causing of the ketolase activity is brought about by anincreasing or causing of the gene expression of a nucleic acid encodinga ketolase comprising the amino acid sequence SEQ ID NO: 2 or a sequencewhich is derived from this sequence by substitution, insertion ordeletion of amino acids and which has an identity of at least 42% at theamino acid level with the sequence SEQ ID NO: 2, compared with the wildtype.
 27. The genetically modified organism as claimed in claim 26,wherein to increase or cause the gene expression nucleic acids whichencode ketolases comprising the amino acid sequence SEQ ID NO: 2 or asequence which is derived from this sequence by substitution, insertionor deletion of amino acids and which has an identity of at least 42% atthe amino acid level with the sequence SEQ ID NO: 2, are introduced intothe organism.
 28. A genetically modified organism comprising at leastone transgenic nucleic acid encoding a ketolase comprising the aminoacid sequence SEQ ID NO: 2 or a sequence which is derived from thissequence by substitution, insertion or deletion of amino acids and whichhas an identity of at least 42% at the amino acid level with thesequence SEQ ID NO:
 2. 29. A genetically modified organism comprising atleast two endogenous nucleic acids encoding a ketolase comprising theamino acid sequence SEQ ID NO: 2 or a sequence which is derived fromthis sequence by substitution, insertion or deletion of amino acids andwhich has an identity of at least 42% at the amino acid level with thesequence SEQ ID NO:
 2. 30. The genetically modified organism as claimedin claim 25, wherein the genetic modification additionally increases atleast one of the activities selected from the group consisting ofhydroxylase activity and β-cyclase activity, compared with the wildtype.
 31. The genetically modified organism as claimed in claim 25,which is able as starting organism naturally or through geneticcomplementation to produce carotenoids.
 32. The genetically modifiedorganism as claimed in claim 25, selected from the group consisting ofmicroorganisms OF and plants.
 33. The genetically modified organism asclaimed in claim 32, wherein the microorganisms are selected from thegroup consisting of bacteria, yeasts, algae of and fungi.
 34. Thegenetically modified microorganism as claimed in claim 33, wherein themicroorganisms are selected from the group consisting of Escherichia,Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Paracoccus, Nostoc,cyanobacteria of the genus Synechocystis, Candida, Saccharomyces,Hansenula, Pichia, Aspergillus, Trichoderma, Ashbya, Neurospora,Blakeslea, Phycomyces, Fusarium, Haematococcus, Phaedactylumtricornatum, Volvox and Dunaliella.
 35. The genetically modified plantas claimed in claim 32, wherein the plants are selected from thefamilies consisting of Ranunculaceae, Berberidaceae, Papaveraceae,Cannabaceae, Rosaceae, Fabaceae, Linaceae, Vitaceae, Brassiceae,Cucurbitaceae, Primulaceae, Caryophyllaceae, Amaranthaceae,Gentianaceae, Geraniaceae, Caprifoliaceae, Oleaceae, Tropaeolaceae,Solanaceae, Scrophulariaceae, Asteraceae, Liliaceae, Amaryllidaceae,Poaceae, Orchidaceae, Malvaceae, Illiaceae and Lamiaceae.
 36. Thegenetically modified plant as claimed in claim 35, wherein the plantsare selected from the plant genera consisting of Marigold, Tageteserecta, Tagetes patula, Acacia, Aconitum, Adonis, Arnica, Aquilegia,Aster, Astragalus, Bignonia, Calendula, Caltha, Campanula, Canna,Centaurea, Cheiranthus, Chrysanthemum, Citrus, Crepis, Crocus,Curcurbita, Cytisus, Delonia, Delphinium, Dianthus, Dimorphotheca,Doronicum, Eschscholtzia, Forsythia, Fremontia, Gazania, Gelsemium,Genista, Gentiana, Geranium, Gerbera, Geum, Grevillea, Helenium,Helianthus, Hepatica, Heracleum, Hibiscus, Heliopsis, Hypericum,Hypochoeris, Impatiens, Iris, Jacaranda, Kerria, Labumum, Lathyrus,Leontodon, Lilium, Linum, Lotus, Lycopersicon, Lysimachia, Maratia,Medicago, Mimulus, Narcissus, Oenothera, Osmanthus, Petunia, Photinia,Physalis, Phyteuma, Potentilla, Pyracantha, Ranunculus, Rhododendron,Rosa, Rudbeckia, Senecio, Silene, Silphium, Sinapsis, Sorbus, Spartium,Tecoma, Torenia, Tragopogon, Trollius, Tropaeolum, Tulipa, Tussilago,Ulex, Viola and Zinnia.
 37. An animal or human food comprising thegenetically modified organism as claimed in claim
 25. 38. A method forproducing ketocarotenoid-containing extracts or for producing animal andhuman food supplements comprising cultivating the genetically modifiedorganism as claimed in claim 25 and recovering ketocarotenoid.
 39. Aketolase comprising the amino acid sequence SEQ. ID. NO. SEQ ID NO: 2 ora sequence which is derived from this sequence by substitution,insertion or deletion of amino acids and which has an identity of atleast 70% at the amino acid level with the sequence SEQ ID NO: 2, withthe proviso that the amino acid sequence SEQ ID NO: 2 is not present.40. A ketolase comprising the amino acid sequence SEQ ID NO: 4 or asequence which is derived from this sequence by substitution, insertionor deletion of amino acids and which has an identity of at least 70% atthe amino acid level with the sequence SEQ ID NO:
 4. 41. A nucleic acidencoding a protein as claimed in claim 39, with the proviso that thesequences SEQ ID NO: 1 and SEQ ID NO: 3 are not present.
 42. (canceled)43. A ketolase comprising the amino acid sequence SEQ ID NO: 4 or asequence which is derived from this sequence by substitution, insertionor deletion of amino acids and which has an identity of at least 65% atthe amino acid level with the sequence SEQ ID NO: 4.